Tropoelastin for promoting endothelial cell adhesion or migration

ABSTRACT

The invention provides methods, compositions, and devices for promoting adhesion or migration of endothelial cell.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/885,105, which is a 371 national stage of International ApplicationNo. PCT/US06/06526 (filed Feb. 24, 2006) which claims priority to U.S.Provisional Application No. 60/656,360, filed Feb. 25, 2005, thecontents of all of which are hereby incorporated by reference in theirentireties.

This invention was made with government support under RO1 HL068873awarded by the National Institutes of Health. The government has certainrights in the invention.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

A sequence listing, created on Jan. 13, 2016 as the ASCII text file“3026-6026-CON_SeqListing_v2.txt” having a file size of 22 kilobytes, isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Advances in the treatment of cardiovascular diseases and obstructivevascular diseases have led to treatments for a range of illnesses andconditions. These treatments, which include implantation of numerousvarieties of drug-eluting and non-drug-eluting devices, havedramatically improved survival and the quality of life of patients.However, these treatments are not without limitations and side-effects.

Medical therapies focus on reducing the risk factors associated withobstructive vascular disease. Anti-thrombotic, anti-hypertensive, andcholesterol-lowering medications are aimed at decreasing the risk ofocclusion, while beta-blockers and angiotensin-converting enzymeinhibitors act by reducing the workload of the heart. Despite thesepharmaceutical advances that reduce the risk of vascular occlusion andcardiac events, the need for interventional cardiology and cardiacsurgery to directly treat cardiovascular diseases and vascularobstructions remains immense.

Angioplasty is the major intervention for coronary artery diseaseaccounting for over 680,000 procedures annually in the United Statesalone. Briefly, a balloon is placed within a blocked artery and expandedto relieve the obstruction. In most cases, a stent is placed within theinstrumented artery. However, only 70% of angioplasties lead tolong-term (6 months) relief of vascular obstruction. In a process termedrestenosis, vascular smooth muscle cells reocclude the artery inresponse to the vascular injury caused by angioplasty. Such restenosisis also observed in other procedures which cause injury to a body vesselwall including the placement of stents, wires, catheters, shunts, orother intraluminal devices in any body vessel (e.g., in an artery, vein,ureter, urethra, Fallopian tube, common bile duct, pancreatic duct,kidney duct, esophagus, trachea, bladder, uterus, ovarian duct, vasdeferens, prostatic duct, or lymphatic duct.). Continued advances in thegeometry and composition of stents have largely impacted ease of stentdelivery, but have not lessened the complication of restenosis.Recently, strategies employing radioactivity and other cytotoxic agents(e.g. paclitaxil, actinomycinD, and rapamycin) to treat restenosis havereceived substantial attention. These strategies rely on the temporaryand local delivery of toxic agents that block proliferation of many celltypes. The long-term efficacy is currently being tested; however thetoxicity of these agents raises serious doubts of whether they can beused as a long term treatment option for managing often chronic cardiacand cardiovascular conditions.

Current therapies for cardiovascular and obstructive vascular diseasesaim to prevent or inhibit the hyperproliferation of the vascular smoothmuscle cells lining vessels, thereby preventing or inhibiting occlusionof the vessel. However, many of the therapies, including rapamycin andtaxol, used to inhibit proliferation of vascular smooth muscle cellsfunction via a cytotoxic mechanism. Although these therapies may helpprevent occlusion, their cytotoxicity inhibits endothelial cells withinor adjacent to vessels. Damage to endothelial cell growth or structureis further exacerbated by insertion of intraluminal devices which areoften used to deliver these and other therapeutic agents. A combinationof the damage caused by the therapeutic agents and/or damage caused bythe insertion of various devices often leads to restenosis. As a resultof restenosis, patients with cardiovascular and occlusive vasculardiseases must often be repeatedly stented, catheterized, or otherwisetreated. Such repeated treatment exposes the patients to increased risksassociated with any hospitalization or invasive procedure. Furthermore,the need to repeat these treatments dramatically increases the costsassociated with managing these conditions. Methods and compositions thatdecrease or prevent restenosis would provide a substantial improvementin the art.

In addition to the problem of restenosis, thrombosis is a significantproblem associated with many of the current treatments forcardiovascular diseases and occlusive vascular diseases. In fact, therisk of thrombosis exists whenever a device is placed and left in thebody, and is thus a potential complication of many surgical procedures.Thus, thrombosis is a serious complication associated not only withcardiac and intravascular procedures, but also with other interventionalapproaches involving the placing of devices into the body or into thelumens of body vessels. Specifically, and as outlined above, many of thecurrent drug therapies damage endothelial cells and then inhibit theirproliferation. This significantly inhibits endothelialization ofinserted devices. As a result, late thrombosis may occur in patientstreated using a drug-eluting or non-drug-eluting device. To help preventpotentially lethal thrombosi, patients are often aggressively treated(sometimes on a long term basis) with anti-platelet and/oranti-coagulant therapies. These treatments impose their own risks andcosts. Accordingly, methods and compositions that promoteendothelization of inserted devices (e.g., intravascular or otherintraluminal devices), thereby preventing or decreasing the likelihoodof thrombosis would be a significant improvement in the art.

The present invention provides methods and compositions for promotingadhesion of endothelial cells, for example, for promoting adhesion ofendothelial cells to implantable, biocompatible devices. Methods andcompositions for promoting adhesion of endothelial cells, for example,to biocompatible devices, can be used in the treatment or prevention ofrestenosis and/or thrombosis.

SUMMARY OF THE INVENTION

The invention provides compositions and methods for modulating theadhesion and/or migration of endothelial cells. The invention providescompositions and methods for treating and preventing restenosis orthrombosis that often result as a potentially fatal side-effect of drugand device-based therapies for numerous cardiovascular diseases andobstructive vascular diseases. The invention further providesimplantable devices designed to promote endothelial adhesion, therebypreventing restenosis or thrombosis.

In a first aspect, the invention provides a method of promoting adhesionof endothelial cells. The method comprises contacting said endothelialcells with an amount of a composition comprising an effective amount ofa tropoelastin polypeptide or bioactive fragment thereof, wherein saideffective amount is sufficient to promote adhesion of endothelial cells.The tropoelastin polypeptide or bioactive fragment thereof, has one ormore of the biological activities of native tropoelastin.

In one embodiment, the method of promoting adhesion of endothelial cellsis an in vitro method. In another embodiment, the method of promotingadhesion of endothelial cells is an in vivo method.

In one embodiment, the tropoelastin polypeptide or bioactive fragmentthereof comprises an amino acid sequence at least 80% identical to SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. Inanother embodiment, the tropoelastin polypeptide, or bioactive fragmentthereof, comprises an amino acid sequence identical to SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In still anotherembodiment, the composition consists essentially of an amino acidsequence at least 8% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, or SEQ ID NO: 6. In yet another embodiment, thecomposition consists essentially of at least one repeat of a bioactivefragment represented in SEQ ID NO: 5. In another embodiment, thecomposition consists essentially of a bioactive fragment represented inSEQ ID NO: 6. In still another embodiment, said tropoelastinpolypeptide, or bioactive fragment thereof, comprises an amino acidsequence encodable by a nucleic acid that hybridizes under stringentconditions, including a wash step of 0.2×SSC at 65° C., to a nucleicacid sequence represented in SEQ ID NO: 1. In any of the foregoing, thecomposition comprises or consists essentially of a tropoelastinpolypeptide, or bioactive fragment thereof, that retains one or more ofthe biological activities of native tropoelastin. Exemplarybiological/functional activities of native tropoelastin that areretained by the tropoelastin polypeptides, or bioactive fragmentsthereof, for use in the methods and devices of the invention include,but are not limited to: (i) promotes adhesion of endothelial cells invitro; (ii) promotes adhesion of endothelial cells in vivo; (iii)promotes adhesion of human aortic endothelial cells (HuAEC) in vitro;(iv) promotes adhesion of human aortic smooth muscle cells (HuAoSMC) invitro; (v) promotes migration of endothelial cells in vitro; (vi)promotes migration of human aortic endothelial cells (HuAEC) in vitro;(vii) promotes adhesion of A2058 human melanoma cells in vitro; (viii)promotes migration of A2058 human melanoma cells in vitro; (ix) promotesadhesion of human microvessel endothelial cells (HMVEC) in vitro.

In another embodiment, the tropoelastin polypeptide, or bioactivefragment thereof, is covalently attached to a device. Exemplary devicesare made of or coated with metal, silicone, dacron, plastic, orpolytetrafluoroethylene (PTFE). Exemplary metal devices includestainless steel devices. An effective amount of the tropoelastinpolypeptide, or bioactive fragment thereof, is attached to the deviceand the effective amount is sufficient to promote adhesion ofendothelial cells to the device.

In a second aspect, the invention promotes a method of promotingmigration of endothelial cells. The method comprises contacting saidendothelial cells with an amount of a composition comprising aneffective amount of a tropoelastin polypeptide, or bioactive fragmentthereof, wherein said effective amount is sufficient to promotemigration of endothelial cells. The tropoelastin polypeptide, orbioactive fragment thereof, has one or more of the biological activitiesof native tropoelastin.

In one embodiment, the method of promoting migration of endothelialcells is an in vitro method. In another embodiment, the method ofpromoting migration of endothelial cells is an in vivo method.

In one embodiment, the tropoelastin polypeptide, or bioactive fragmentthereof, comprises an amino acid sequence at least 80% identical to SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. Inanother embodiment, the tropoelastin polypeptide, or bioactive fragmentthereof, comprises an amino acid sequence identical to SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In still anotherembodiment, the composition consists essentially of an amino acidsequence at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In yet another embodiment, thecomposition consists essentially of at least one repeat of a bioactivefragment represented in SEQ ID NO: 5. In another embodiment, thecomposition consists essentially of a bioactive fragment represented inSEQ ID NO: 6. In still another embodiment, said tropoelastinpolypeptide, or bioactive fragment thereof, comprises an amino acidsequence encodable by a nucleic acid that hybridizes under stringentconditions, including a wash step of 0.2×SSC at 65° C., to a nucleicacid sequence represented in SEQ ID NO: 1. In any of the foregoing, thecomposition comprises or consists essentially of a tropoelastinpolypeptide, or bioactive fragment thereof, that retains one or more ofthe biological activities of native tropoelastin. Exemplarybiological/functional activities of native tropoelastin that areretained by the tropoelastin polypeptides, or bioactive fragmentsthereof, for use in the methods and devices of the invention include,but are not limited to: (i) promotes adhesion of endothelial cells invitro; (ii) promotes adhesion of endothelial cells in vivo; (iii)promotes adhesion of human aortic endothelial cells (HuAEC) in vitro;(iv) promotes adhesion of human aortic smooth muscle cells (HuAoSMC) invitro; (v) promotes migration of endothelial cells in vitro; (vi)promotes migration of human aortic endothelial cells (HuAEC) in vitro;(vii) promotes adhesion of A2058 human melanoma cells in vitro; (viii)promotes migration of A2058 human melanoma cells in vitro; (ix) promotesadhesion of human microvessel endothelial cells (HMVEC) in vitro.

In another embodiment, the tropoelastin polypeptide, or bioactivefragment thereof, is covalently attached to a device. Exemplary devicesare made of or coated with metal, plastic, Dacron, PTFE, silicone, orplastic. Exemplary metal devices include stainless steel devices. Aneffective amount of the tropoelastin polypeptide, or bioactive fragmentthereof, is attached to the device and the effective amount issufficient to promote migration of endothelial cells to the device.

In a third aspect, the invention provides a method of promoting adhesionof endothelial cells to a device. The method comprises contacting saidendothelial cells with a device. The device comprises a compositioncomprising an effective amount of a tropoelastin polypeptide, orbioactive fragment thereof, and the tropoelastin or bioactive fragmentthereof is covalently attached to the device. An effective amount of thetropoelastin polypeptide, or bioactive fragment thereof, is attached tothe device and the effective amount is sufficient to promote adhesion ofendothelial cells to the device.

In one embodiment, the method is an in vitro method. In anotherembodiment, the method is an in vivo method.

In one embodiment, the device is a metal device, for example, astainless steel device. In another embodiment, the device is made fromor coated with plastic, silicone, PTFE, or dacron. In one embodiment,the device is selected from a catheter, stent, shunt, wire, or otherintraluminal device. In another embodiment, the device is selected froma pacemaker, cardioverter-defibrillator, artificial valve, ventricularassist device, vascular graft, nasogastric tube, ventilator tube, orchest tube.

In one embodiment, the tropoelastin polypeptide, or bioactive fragmentthereof, comprises an amino acid sequence at least 80% identical to SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. Inanother embodiment, the tropoelastin polypeptide, or bioactive fragmentthereof, comprises an amino acid sequence identical to SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In still anotherembodiment, the composition consists essentially of an amino acidsequence at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In yet another embodiment, thecomposition consists essentially of at least one repeat of a bioactivefragment represented in SEQ ID NO: 5. In another embodiment, thecomposition consists essentially of a bioactive fragment represented inSEQ ID NO: 6. In still another embodiment, said tropoelastinpolypeptide, or bioactive fragment thereof, comprises an amino acidsequence encodable by a nucleic acid that hybridizes under stringentconditions, including a wash step of 0.2×SSC at 65° C., to a nucleicacid sequence represented in SEQ ID NO: 1. In any of the foregoing, thecomposition comprises or consists essentially of a tropoelastinpolypeptide, or bioactive fragment thereof, that retains one or more ofthe biological activities of native tropoelastin. Exemplarybiological/functional activities of native tropoelastin that areretained by the tropoelastin polypeptides, or bioactive fragmentsthereof, for use in the methods and devices of the invention include,but are not limited to: (i) promotes adhesion of endothelial cells invitro; (ii) promotes adhesion of endothelial cells in vivo; (iii)promotes adhesion of human aortic endothelial cells (HuAEC) in vitro;(iv) promotes adhesion of human aortic smooth muscle cells (HuAoSMC) invitro; (v) promotes migration of endothelial cells in vitro; (vi)promotes migration of human aortic endothelial cells (HuAEC) in vitro;(vii) promotes adhesion of A2058 human melanoma cells in vitro; (viii)promotes migration of A2058 human melanoma cells in vitro; (ix) promotesadhesion of human microvessel endothelial cells (HMVEC) in vitro.

In a fourth aspect, the invention provides a method for the treatment orprophylaxis of restenosis. The method comprises administering an amountof a composition comprising an amount of tropoelastin, or a bioactivefragment thereof, effective to treat or prophylactically treatrestenosis. The tropoelastin, or bioactive fragment thereof, iscovalently attached to a device, and administering said compositionpromotes adhesion of endothelial cells to said device, thereby treatingor preventing restenosis.

In one embodiment, the method is performed following angioplasty,catheterization, stenting, surgery, or other interventional therapy. Inanother embodiment, the method is performed concomitantly withangioplasty, catheterization, stenting, surgery, or other interventionaltherapy. In still another embodiment, the same device used to preventthe occlusion is also coated according to the invention in order toprevent restenosis.

In one embodiment, one or more other agents are concurrently orconcomitantly administered as part of a treatment regimen appropriatefor the particular cardiovascular or occlusive vessel condition.

In one embodiment, the device is selected from a stent, catheter, shunt,wire, or other intraluminal device. In one embodiment, the device is ametal device, for example, a stainless steel device.

In one embodiment, the method comprises administering said deviceintravascularly or intraluminally to a site of cell damage within a bodyvessel. In another embodiment, the body vessel is selected from any ofartery, vein, ureter, common bile duct, pancreatic duct, kidney duct,esophagus, trachea, urethra, bladder, uterus, ovarian duct, Fallopiantube, yes deferens, prostatic duct, or lymphatic duct.

In one embodiment, the tropoelastin polypeptide, or bioactive fragmentthereof, comprises an amino acid sequence at least 80% identical to SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. Inanother embodiment, the tropoelastin polypeptide, or bioactive fragmentthereof, comprises an amino acid sequence identical to SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In still anotherembodiment, the composition consists essentially of an amino acidsequence at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In yet another embodiment, thecomposition consists essentially of at least one repeat of a bioactivefragment represented in SEQ ID NO: 5. In another embodiment, thecomposition consists essentially of a bioactive fragment represented inSEQ ID NO: 6. In still another embodiment, said tropoelastinpolypeptide, or bioactive fragment thereof, comprises an amino acidsequence encodable by a nucleic acid that hybridizes under stringentconditions, including a wash step of 0.2×SSC at 65° C., to a nucleicacid sequence represented in SEQ ID NO: 1. In any of the foregoing, thecomposition comprises or consists essentially of a tropoelastinpolypeptide, or bioactive fragment thereof, that retains one or more ofthe biological activities of native tropoelastin. Exemplarybiological/functional activities of native tropoelastin that areretained by the tropoelastin polypeptides, or bioactive fragmentsthereof, for use in the methods and devices of the invention include,but are not limited to: (i) promotes adhesion of endothelial cells invitro; (ii) promotes adhesion of endothelial cells in vivo; (iii)promotes adhesion of human aortic endothelial cells (HuAEC) in vitro;(iv) promotes adhesion of human aortic smooth muscle cells (HuAoSMC) invitro; (v) promotes migration of endothelial cells in vitro; (vi)promotes migration of human aortic endothelial cells (HuAEC) in vitro;(vii) promotes adhesion of A2058 human melanoma cells in vitro; (viii)promotes migration of A2058 human melanoma cells in vitro; (ix) promotesadhesion of human microvessel endothelial cells (HMVEC) in vitro.

In a fifth aspect, the invention provides a method for the treatment orprophylaxis of thrombosis. The method comprises administering an amountof a composition comprising an amount of tropoelastin, or a bioactivefragment thereof, effective to treat or prophylactically treatthrombosis. The tropoelastin, or bioactive fragment thereof, iscovalently attached to a device, and administering said compositionpromotes adhesion of endothelial cells to said device, thereby treatingor preventing thrombosis.

In one embodiment, the method is performed following angioplasty,catheterization, stenting, surgery, or other interventional therapy. Inanother embodiment, the method is performed concomitantly toangioplasty, catheterization, stenting, or other interventional therapy.In still another embodiment, the same device used to prevent theocclusion is also coated according to the invention to preventthrombosis.

In one embodiment, one or more other agents are concurrently orconcomitantly administered as part of a treatment regimen appropriatefor the particular cardiovascular or occlusive vessel condition.

In one embodiment, the device is selected from a stent, catheter, shunt,wire, or other intraluminal device. In another embodiment, the device isselected from a pacemaker, cardioverter-defibrillator, artificial valve,ventricular assist device, vascular graft, nasogastric device,ventilator tube, or chest tube. In one embodiment, the device is a metaldevice. In another embodiment, the metal device is a stainless steeldevice. In another embodiment, the device is made of or coated withplastic, silicone, Dacron, or PTFE.

In one embodiment, the method comprises administering said deviceintravascularly or intraluminally to a site of cell damage within a bodyvessel. In another embodiment, the body vessel is selected from any ofartery, vein, ureter, common bile duct, pancreatic duct, kidney duct,esophagus, trachea, urethra, bladder, uterus, ovarian duct, Fallopiantube, vas deferens, prostatic duct, or lymphatic duct.

In one embodiment, the tropoelastin polypeptide, or bioactive fragmentthereof, comprises an amino acid sequence at least 80% identical to SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. Inanother embodiment, the tropoelastin polypeptide, or bioactive fragmentthereof, comprises an amino acid sequence identical to SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In still anotherembodiment, the composition consists essentially of an amino acidsequence at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In yet another embodiment, thecomposition consists essentially of at least one repeat of a bioactivefragment represented in SEQ ID NO: 5. In another embodiment, thecomposition consists essentially of a bioactive fragment represented inSEQ ID NO: 6. In still another embodiment, said tropoelastinpolypeptide, or bioactive fragment thereof, comprises an amino acidsequence encodable by a nucleic acid that hybridizes under stringentconditions, including a wash step of 0.2×SSC at 65° C., to a nucleicacid sequence represented in SEQ ID NO: 1. In any of the foregoing, thecomposition comprises or consists essentially of a tropoelastinpolypeptide, or bioactive fragment thereof, that retains one or more ofthe biological activities of native tropoelastin. Exemplarybiological/functional activities of native tropoelastin that areretained by the tropoelastin polypeptides, or bioactive fragmentsthereof, for use in the methods and devices of the invention include,but are not limited to: (i) promotes adhesion of endothelial cells invitro; (ii) promotes adhesion of endothelial cells in vivo; (iii)promotes adhesion of human aortic endothelial cells (HuAEC) in vitro;(iv) promotes adhesion of human aortic smooth muscle cells (HuAoSMC) invitro; (v) promotes migration of endothelial cells in vitro; (vi)promotes migration of human aortic endothelial cells (HuAEC) in vitro;(vii) promotes adhesion of A2058 human melanoma cells in vitro; (viii)promotes migration of A2058 human melanoma cells in vitro; (ix) promotesadhesion of human microvessel endothelial cells (HMVEC) in vitro.

In a sixth aspect, the invention provides a device. The device comprisesa tropoelastin polypeptide, or a bioactive fragment thereof. Thetropoelastin polypeptide, or bioactive fragment thereof, is covalentlyattached to said device. The device comprises an amount of saidtropoelastin polypeptide, or bioactive fragment thereof, effective topromote adhesion of endothelial cells to said device.

In one embodiment, the device is a metal device. In another embodiment,the metal device is a stainless steel device. In another embodiment, thedevice is selected from a catheter, stent, shunt, wire, or otherintraluminal device. In another embodiment, the device is selected froma pacemaker, cardioverter-defibrillator, artificial valve, ventricularassist device, vascular graft, nasogastric tube, ventilator tube, orchest tube.

In a seventh aspect, the invention provides a method for screening toidentify bioactive fragments of tropoelastin that retain one or more ofthe biological activities of tropoelastin. In one embodiment, thescreening method is performed using endothelial cells or endothelialprogenitor cells in culture to identify and/or characterize bioactivefragments of tropoelastin that retain the ability to promote adhesionand/or migration of endothelial cells in vitro.

In one embodiment, the endothelial cells in culture are mammalian cells.In another embodiment, the endothelial cells in culture are mouse cellsor rat cells. In another embodiment, the endothelial cells in cultureare pig cells. In still another embodiment, the endothelial cells inculture are human cells. In another embodiment, the endothelial cellsare human microvessel endothelial cells. In yet another embodiment, theendothelial cells are human aortic endothelial cells (HuAEC).

In one embodiment, the screening method is a high-throughput screeningmethod.

The invention contemplates combinations of any of the foregoing aspectsand embodiments. In one embodiment of any of the foregoing, theinvention provides a method of promoting adhesion of endothelial cells.In one embodiment, the invention provides a method of promoting adhesionof endothelial stem cells.

In certain embodiments of any of the foregoing, the invention providesmethods for promoting adhesion of endothelial cells or endothelial stemcells, for example, methods of promoting adhesion to a device. Theinvention further provides devices to which a tropoelastin polypeptideor bioactive fragment is covalently attached or appended. In certainembodiments, the adhesion of endothelial cells or endothelial stem cellsis sufficient such that adherent cells are retained on the device underphysiologically relevant flow rates and/or pressures. By physiologicallyrelevant is meant that cells that adhere to a device bearingtropoelastin or a bioactive fragment thereof are retained on the deviceat flow rates and/or pressures equivalent to venous and/or arterial flowrates and/or pressures. For embodiments in which a device is placeintraluminally into a vessel other than an artery or vein, the termphysiologically relevant refers to the pressure and/or fluid flow ratesequivalent to those experienced in the particular vessel (e.g., vasdeferens, urethra, ureter, prostatic duct, bile duct, etc.).

In any of the foregoing, tropoelastin or bioactive fragments thereof maybe used alone to promote adhesion and/or migration of endothelial cells.Alternatively, tropoelastin or bioactive fragments thereof may be usedin combination with one or more other therapeutic regimens appropriateto the particular condition being treated. Furthermore, tropoelastin, orbioactive fragments thereof, may be used alone or in combination withanother adhesion promoting agent. In one embodiment, adhesion ofendothelial cells is promoted using a device coated with tropoelastin,or a bioactive fragment thereof. In another embodiment, the device isco-coated with both tropoelastin, or a bioactive fragment thereof, andanti-CD34 antibody.

In any of the foregoing, devices of the invention to which tropoelastinor one or more bioactive fragments thereof are attached can be made ofor coated with metal, plastic, silicone, dacron, polyurethane,polypropylene, PTFE, or derivatives thereof. Attachment can becovalently, for example via a polysaccharide linkage. Alternatively,attachment can be via crosslinking using commonly employed crosslinkingagents.

In certain embodiments of any of the foregoing, the methods,compositions, and devices of the invention can be used in theprophylaxis or treatment of thrombosis. In certain embodiments, devicescoated with the subject polypeptides may be non-thrombogenic. In otherembodiments, devices coated with the subject polypeptides may beanti-thrombogenic. In still other embodiments, the devices coated withthe subject polypeptides will be substantially less thrombogenic thansimilar uncoated devices. Regardless of whether a particular coateddevice has anti-thrombogenic properties or is simply non-thrombogenic,coated devices according to the present invention decrease or preventthrombosis in comparison to alternative interventional therapies.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, virology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are described in the literature. See, for example,Molecular Cloning: A Laboratory Manual, 3rd Ed., ed. by Sambrook andRussell (Cold Spring Harbor Laboratory Press: 2001); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Using Antibodies,Second Edition by Harlow and Lane, Cold Spring Harbor Press, New York,1999; Current Protocols in Cell Biology, ed. by Bonifacino, Dasso,Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons, Inc., NewYork, 1999.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of fibronectin and tropoelastin on the adhesionof CHO cells, human embryonic kidney cells (HEK 293), human aorticsmooth muscle cells (HuAoSMC), and human aortic endothelial cells(HuAEC). For each cell type, cell adhesion was measured in culturedishes coated with either fibronectin or recombinant human tropoelastin.Note that endothelial cells, and to a lesser extent smooth muscle cells,adhere to culture dishes coated with tropoelastin. In comparison, CHOcells and HEK 293 cells adhere to culture dishes coated with fibronectinbut not to culture dishes coated with tropoelastin.

FIG. 2 graphically summarizes the adhesion of endothelial cells toculture dishes coated with a variety of proteins. The adherence ofHuAECs to tropoelastin is similar to that observed with fibronectin andcollagen.

FIG. 3 depicts the effect of tropoelastin, or various bioactivefragments of tropoelastin, on endothelial cell migration. Tropoelastin,as well as two different bioactive fragments of tropoelastin, promotemigration of endothelial cells. The effect on endothelial cell migrationof tropoelastin, and various bioactive fragments, is comparable to theeffect of fibronectin on endothelial cell migration.

FIG. 4 shows that tropoelastin, and a bioactive fragment oftropoelastin, promote adhesion of A2058 human melanoma cells.

FIG. 5 shows that tropoelastin, and various bioactive fragments oftropoelastin, promote migration of A2058 human melanoma cells.

FIG. 6 shows that another human endothelial cell type (human microvesselendothelial cells—HMVEC) adhered to stainless steel disks to whichtropoelastin was covalently attached. Adhesion of the HMVECs to thetropoelastin coated stainless steel disks was comparable to the adhesionof HMVECs to stainless steel disks coated with an anti-CD34 antibody.

FIG. 7 shows that endothelial progenitor cells adhere to stainless steeldisks that have been coated with either an anti-CD34 antibody or withtropoelastin. However, examination of the cells following their bindingto the stainless steel disks indicated that adhesion to tropoelastin isassociated with cell spreading across the surface of the disk. Incontrast, we observed adhesion but not cell spreading of endothelialcells cultured with stainless steel disks coated with an anti-CD34antibody.

DETAILED DESCRIPTION OF THE INVENTION (i) Overview

Biological properties of elastin and the elastin-signaling pathway areprovided in Li at al. (1998) Journal of Clinical Investigation 102:1783-1787; Li et al. (1998) Nature 393: 276-280; Karnik et al. (2003)Matrix Biology 22: 409-425; Karnik et al. (2003) Development 130:411-423; and Brooke at al. (2003) Trends in Cardiovascular Medicine 13:176-181, the disclosures of which are hereby incorporated by referencein their entirety. Briefly, these and other references demonstrated theeffect of modulating elastin signaling on smooth muscle cells andvascular smooth muscle cells. However, until now, the utility oftropoelastin, and bioactive fragments thereof, to modulate the adhesionand migration of endothelial cells was not recognized.

Based on the findings disclosed in the present application,tropoelastin, and bioactive fragments thereof, can be used to promotethe adhesion and/or migration of endothelial cells. This finding allowsthe development of in vitro methods of using tropoelastin, or bioactivefragments thereof, either alone or in association with a device.Exemplary in vitro uses include screening assays to identify and/orcharacterize fragments of tropoelastin or peptidomimetics that retainone or more of the functional activities of native (e.g., full length)tropoelastin.

This finding also allows for the development of in vivo methods of usingtropoelastin, or bioactive fragments thereof. An exemplary in vivo useis to promote the adhesion of endothelial cells to a device to whichtropoelastin, or a bioactive fragment thereof has been covalentlyattached. In an in vivo context, endothelization of implantable devices(e.g., prior to or following placement in a patient) can help prevent orreduce restenosis and/or thrombosis. The ability to promoteendothelialization of implantable devices is a significant advance thatwill increase the potential efficacy of any of a number ofinterventional approaches. Given that currently used and contemplatedimplantable devices and interventional approaches pose a significantrisk of thrombosis and/or restenosis, the methods and compositions ofthe present invention will substantially reduce the risks involved withthese procedures, thereby increasing their safety and efficacy.

(ii) Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, “protein” is a polymer consisting essentially of any ofthe 20 amino acids. Although “polypeptide” is often used in reference torelatively large polypeptides, and “peptide” is often used in referenceto small polypeptides, usage of these terms in the art overlaps and isvaried.

The terms “peptide(s)”, “protein(s)” and “polypeptide(s)” are usedinterchangeably herein.

The terms “polynucleotide sequence” and “nucleotide sequence” are alsoused interchangeably herein.

“Recombinant,” as used herein, means that a protein is derived from aprokaryotic or eukaryotic expression system.

The term “wildtype” refers to the naturally-occurring polynucleotidesequence encoding a protein, or a portion thereof, or protein sequence,or portion thereof, respectively, as it normally exists in vivo.

The term “mutant” refers to any change in the genetic material of anorganism, in particular a change (i.e., deletion, substitution,addition, or alteration) in a wild type polynucleotide sequence or anychange in a wild type protein. The term “variant” is usedinterchangeably with “mutant”. Although it is often assumed that achange in the genetic material results in a change of the function ofthe protein, the terms “mutant” and “variant” refer to a change in thesequence of a wildtype protein regardless of whether that change altersthe function of the protein (e.g., increases, decreases, imparts a newfunction), or whether that change has no effect on the function of theprotein (e.g., the mutation or variation is silent).

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of RNA or DNA made from nucleotide analogs, and, as applicableto the embodiment being described, single (sense or antisense) anddouble-stranded polynucleotides.

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding a polypeptide,including both exon and (optionally) intron sequences.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. Preferred vectors are those capable of autonomous replicationand/or expression of nucleic acids to which they axe linked. Vectorscapable of directing the expression of genes to which they areoperatively linked are referred to herein as “expression vectors”.

A polynucleotide sequence (DNA, RNA) is “operatively linked” to anexpression control sequence when the expression control sequencecontrols and regulates the transcription and translation of thatpolynucleotide sequence. The term “operatively linked” includes havingan appropriate start signal (e.g., ATG) in front of the polynucleotidesequence to be expressed, and maintaining the correct reading frame topermit expression of the polynucleotide sequence under the control ofthe expression control sequence, and production of the desiredpolypeptide encoded by the polynucleotide sequence.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to nucleic acid sequences, such as initiationsignals, enhancers, and promoters, which induce or control transcriptionof protein coding sequences with which they are operably linked. In someexamples, transcription of a recombinant gene is under the control of apromoter sequence (or other transcriptional regulatory sequence) whichcontrols the expression of the recombinant gene in a cell-type in whichexpression is intended. It will also be understood that the recombinantgene can be under the control of transcriptional regulatory sequenceswhich are the same or which are different from those sequences whichcontrol transcription of the naturally-occurring form of a protein.

As used herein, the term “tissue-specific promoter” means a nucleic acidsequence that serves as a promoter, i.e., regulates expression of aselected nucleic acid sequence operably linked to the promoter, andwhich affects expression of the selected nucleic acid sequence inspecific cells of a tissue, such as cells of neural origin, e.g.neuronal cells. The term also covers so-called “leaky” promoters, whichregulate expression of a selected nucleic acid primarily in one tissue,but cause expression in other tissues as well.

“Identity” refers to sequence similarity between two peptides or betweentwo nucleic acid molecules. Identity can be determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame base or amino acid, then the molecules are identical at thatposition. A degree of identity between sequences is a function of thenumber of matching positions shared by the sequences.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding a polypeptide with a second amino acid sequencedefining a domain (e.g. polypeptide portion) foreign to and notsubstantially homologous with any domain of the first polypeptide. Achimeric protein may present a foreign domain which is found (albeit ina different protein) in an organism which also expresses the firstprotein, or it may be an “interspecies”, “intergenic”, etc. fusion ofprotein structures expressed by different kinds of organisms.

The “non-human animals” of the invention include mammals such as rats,mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs, or RNAs,respectively, that are present in the natural source of themacromolecule. The term isolated as used herein also refers to a nucleicacid or peptide that is substantially free of cellular material orculture medium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments whichare not naturally occurring as fragments and would not be found in thenatural state.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intravascular, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe animal's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The phrase “effective amount” as used herein means that the amount ofone or more agent, material, or composition comprising one or moreagents as described herein which is effective for producing some desiredeffect in a subject.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation.

Functional equivalents of a polypeptide, a polypeptide fragment, or avariant polypeptide are those polypeptides that retain a biologicaland/or an immunological activity of the native or naturally-occurringpolypeptide. Immunological activity refers to the ability to induce theproduction of an antibody against an antigenic epitope possessed by anative polypeptide; biological activity refers to a function, eitherinhibitory or stimulatory, caused by the particular native polypeptidethat excludes immunological activity. In the context of the presentinvention, exemplary biological activities include, but are not limitedto: (i) promotes adhesion of endothelial cells in vitro; (ii) promotesadhesion of endothelial cells in vivo; (iii) promotes adhesion of humanaortic endothelial cells (HuAEC) in vitro; (iv) promotes adhesion ofhuman aortic smooth muscle cells (HuAoSMC) in vitro; (v) promotesmigration of endothelial cells in vitro; (vi) promotes migration ofhuman aortic endothelial cells (HuAEC) in vitro; (vii) promotes adhesionof A2058 human melanoma cells in vitro; (viii) promotes migration ofA2058 human melanoma cells in vitro; (ix) promotes adhesion of humanmicrovessel endothelial cells (HMVEC) in vitro. Further exemplarybiological activities include the ability to bind to a particularreceptor, the ability to activate transcription of a particular gene,the ability to inhibit transcription of a particular gene, the abilityto associate (e.g., directly or indirectly associate) with a particularcofactor, the ability to promote signaling via a particular signaltransduction pathway, and the ability to inhibit signaling via anotherparticular signal transduction pathway.

Variant nucleic acid or amino acid sequences may be full length or otherthan full length. Variants of the nucleic acids or proteins of theinvention include, but are not limited to, molecules comprising regionsthat are identical to the nucleic acids or proteins of the invention. Invarious embodiments, the variants are at least about 60%, 65%, 70%, 75%,80%, 83%, 85%, 90%, 95%, 97%, 98%, 99%, or greater than 99% identical toa nucleic acid or amino acid sequence of identical size or when comparedto an aligned sequence in which the alignment is done by a computerhomology program known in the art, or whose encoding nucleic acid iscapable of hybridizing to the complement of a sequence encoding theaforementioned proteins under stringent, moderately stringent, or lowstringent conditions. Variants can also be described with respect to howmany residues differ between polypeptides. For example, a variant maydiffer from a given polypeptide sequence at one of six amino acidresidues, at two of six amino acid residues, or at three of six aminoacid residues.

A “homologous nucleic acid sequence” or “homologous amino acidsequence,” or variations thereof, refer to sequences characterized by adegree of identity at the nucleotide level or amino acid level asdiscussed above. Homologous nucleotide sequences encode those sequencescoding for isoforms of a particular sequence. Isoforms can be expressedin different tissues of the same organism as a result of, for example,alternative splicing of RNA. Alternatively, different genes can encodeisoforms. Homologous nucleotide sequences include nucleotide sequencesencoding a polypeptide from other species, including, but not limitedto: vertebrates, and thus can include, e.g., human, frog, mouse, rat,rabbit, dog, cat, cow, horse, and other organisms. Homologous nucleotidesequences also include, but are not limited to, naturally occurringallelic variations and mutations of the nucleotide sequences set forthherein. A homologous nucleotide sequence does not, however, include theexact nucleotide sequence encoding a particular protein. Homologousnucleic acid sequences include those nucleic acid sequences that encodeconservative amino acid substitutions (see below).

As used herein, certain exemplary compositions comprise, consist of; orconsist essentially of all or a portion of a tropoelastin amino acid ornucleic acid sequence. As used herein, “tropoelastin” refers to all or aportion of a protein encodable by all or a portion of an elastin gene.The term also refers to splice and proteolytic products of an elastingene. The term also refers to tropoelastin protein and protein fragmentswhether encoded by an elastin gene, synthetically produced, or purifiedfrom a naturally occurring human or animal source of elastin. The termtropoelastin, or bioactive fragment thereof, will be used throughout theapplication to refer to any of the foregoing elastin-based compositions.An exemplary nucleic acid sequence of a human tropoelastin isrepresented in SEQ ID NO: 1 and an amino acid sequence is represented inSEQ ID NO: 2. Further exemplary tropoelastin amino acid sequences arerepresented in SEQ ID NO: 3 and 4. Tropoelastin is able to stimulateelastin signaling, and stimulation of elastin signaling has one or moreof the following functional consequences including, but not limited to:(i) promotes adhesion of endothelial cells in vitro; (ii) promotesadhesion of endothelial cells in vivo; (iii) promotes adhesion of humanaortic endothelial cells (HuAEC) in vitro; (iv) promotes adhesion ofhuman aortic smooth muscle cells (HuAoSMC) in vitro; (v) promotesmigration of endothelial cells in vitro; (vi) promotes migration ofhuman aortic endothelial cells (HuAEC) in vitro; (vii) promotes adhesionof A2058 human melanoma cells in vitro; (viii) promotes migration ofA2058 human melanoma cells in vitro; (ix) promotes adhesion of humanmicrovessel endothelial cells (HMVEC) in vitro. Tropoelastinpolypeptides, or bioactive fragments thereof, for use in the methods anddevices of the present invention possess one or more of the foregoingfunctional activities of native tropoelastin. In one embodiment, theadhesion promoted by tropoelastin, or a bioactive fragment, is dependant(in whole or in part) on integrin α_(v)β₃.

In addition to full length tropoelastin, bioactive fragments oftropoelastin can stimulate elastin signaling. By bioactive fragment ismeant that a given portion of the protein maintains one or more of thefunctional attributes of the full length protein. In the context of thepresent invention, a bioactive fragment of tropoelastin retains all or aportion of the ability to promote elastin signaling and thus results inone or more of the functional consequences of elastin signalingincluding, but not limited to: (i) promotes adhesion of endothelialcells in vitro; (ii) promotes adhesion of endothelial cells in vivo;(iii) promotes adhesion of human aortic endothelial cells (HuAEC) invitro; (iv) promotes adhesion of human aortic smooth muscle cells(HuAoSMC) in vitro; (v) promotes migration of endothelial cells invitro; (vi) promotes migration of human aortic endothelial cells (HuAEC)in vitro; (vii) promotes adhesion of A2058 human melanoma cells invitro; (viii) promotes migration of A2058 human melanoma cells in vitro;(ix) promotes adhesion of human microvessel endothelial cells (HMVEC) invitro. An exemplary bioactive fragment of tropoelastin is provided inSEQ ID NO: 5. Another exemplary bioactive fragment of tropoelastin isprovided in SEQ ID NO: 6. Fragments can be made by any method includingsynthetic methods. The invention contemplates the use not only ofbioactive peptide fragments of tropoelastin, but also peptidomimetics(modified fragments). Exemplary peptidomimetics of tropoelastin arepeptidomimetics of SEQ ID NO: 5 or SEQ ID NO: 6. In one embodiment, theadhesion promoted by tropoelastin, or a bioactive fragment, is dependant(in whole or in part) on integrin α_(v)β₃.

The terms “antiproliferative agent” and “antiproliferative compound” areused interchangeably throughout the application to refer to any peptideor nonpeptide agent that inhibit proliferation. The present inventioncontemplates that the compositions and devices of the invention maysometimes be administered concurrently or concomitantly with otheragents as part of a therapeutic treatment regimen appropriate for theparticular indication being treated. One such class of agents comprisesantiproliferative agents. Exemplary antiproliferative agents include,but are not limited to halogenated purine or pyrimidine analogs (e.g.,fluorinated analogs), macrolids, cytoskeletal/microtubule destabilizers,radioactivity, and the like. Further non-limiting examples, ofantiproliferative agents include 5-fluorouracil (5-FU);5′-deoxy-5-fluorouridine; 5-fluorouridine, 2′-deoxy-5-fluorouridine;fluorocytosine; 5-trifluoromethyl-2′-deoxyuridine; alitretinoin(9-cis-retinoic acid); amifostine; bexarotene(4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)ethenyl]benzoicacid); bleomycin; capecitabine (5′-deoxy-5-fluoro-cytidine);chlorambucil; cladribine; cytarabine; daunorubicin; docetaxel;doxorubicin; epirubicin; estramustine; etoposide; exemestane(6-methylenandrosta-1,4-diene-3,17-dione); fludarabine; 5-fluorouracil;gemcitabine; hydroxyurea; idarubicin; irinotecan; melphalan;methotrexate; mitoxantrone; paclitaxel; pentostatin; prednimustine;streptozocin; temozolamide; teniposide; tomudex; topotecan; valrubicin(N-trifluoroacetyladriamycin-14-valerate); vinorelbine; and salts of theforegoing. Still additional examples include actinomycinD; cytochalasinD; dexamethasone; everolimus; sirolimus; tacrolimus; latrunculin A;rapamycin, and analogues thereof such as ABT-578; carvedilol; FK506;taxol; cyclosporine; camptothecin; carubicin; chlorozotocin,chromomycins, including chromomycin A₃, cladribine; colchicine,combretastatin, demecolcine, denopterin, doxorubicin; dromostanolone,edatrexate, enocitabine, epitiostanol, formestane, lentinan, lonidamine,melengestrol, menogaril, nogalamycin; nordihydroguaiaretic acid,olivomycins such as olivomycin A, pirarubicin, plicamycin, porfiromycin,prednimustine, puromycin; ranimustine, ristocetins such as ristocetin A;tegafur, vinblastine, vindesine, and zorubicin. For any of the foregoingantiproliferative agents, the invention similarly contemplates the useof pharmaceutically acceptable salts, esters, prodrugs, analogues andprotected forms thereof which retain all or a portion of theantiproliferative activity. Another class of agents include dietarysupplements. Still another class of agents include beta-blockers andother high blood pressure mediation. The invention contemplatescombinatorial therapies comprising both the compositions and devices ofthe invention along with other therapeutic regimens appropriate to theparticular condition being treated. However, in certain embodiments, theuse of the compositions and devices of the invention obviates the needfor all or a portion of the previously recommended or prescribedtreatment regimen.

The term “appended” refers to the addition of one or more moieties to anamino acid residue. The term refers, without limitation, to the additionof any moiety to any amino acid residue. The term includes attachment ofa moiety via covalent or non-covalent interactions.

The term “N-terminal amino acid residue” refers to the first amino acidresidue (amino acid number 1) of a polypeptide or peptide.

The term “C-terminal amino acid residue” refers to the last amino acidresidue (amino acid number n, wherein n=the total number of residues inthe peptide or polypeptide) of a polypeptide or peptide.

The term “amino acid side chain” is that part of an amino acid exclusiveof the —CH(NH₂)COOH portion, as defined by K. D. Kopple, “Peptides andAmino Acids”, W. A. Benjamin Inc., New York and Amsterdam, 1966, pages 2and 33; examples of such side chains of the common amino acids are—CH₂CH₂SCH₃ (the side chain of methionine), —CH₂(CH₃)—CH₂CH₃ (the sidechain of isoleucine), —CH₂CH(CH₃)₂ (the side chain of leucine) or H—(the side chain of glycine).

In certain embodiments, the amino acids used in the application of thisinvention are those naturally occurring amino acids found in proteins,or the naturally occurring anabolic or catabolic products of such aminoacids which contain amino and carboxyl groups. Particularly suitableamino acid side chains include side chains selected from those of thefollowing amino acids: glycine, alanine, valine, cysteine, leucine,isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid,glutamine, asparagine, lysine, arginine, proline, histidine,phenylalanine, tyrosine, and tryptophan.

The term “amino acid residue” further includes analogs, derivatives andcongeners of any specific amino acid referred to herein, as well asC-terminal or N-terminal protected amino acid derivatives (e.g. modifiedwith an N-terminal or C-terminal protecting group). For example, thepresent invention contemplates the use of amino acid analogs wherein aside chain is lengthened or shortened while still providing a carboxyl,amino or other reactive precursor functional group for cyclization, aswell as amino acid analogs having variant side chains with appropriatefunctional groups). For instance, the subject compound can include anamino acid analog such as, for example, cyanoalanine, canavanine,djenkolic acid, norleucine, 3-phosphoserine, homoserine,dihydroxy-phenylalanine, 5-hydroxytryptophan, 1-methylhistidine,3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyricacid. Other naturally occurring amino acid metabolites or precursorshaving side chains which are suitable herein will be recognized by thoseskilled in the art and are included in the scope of the presentinvention.

Also included are the (D) and (L) stereoisomers of such amino acids whenthe structure of the amino acid admits of stereoisomeric forms. Theconfiguration of the amino acids and amino acid residues herein aredesignated by the appropriate symbols (D), (L) or (DL), furthermore whenthe configuration is not designated the amino acid or residue can havethe configuration (D), (L) or (DL). It will be noted that the structureof some of the compounds of this invention includes asymmetric carbonatoms. It is to be understood accordingly that the isomers arising fromsuch asymmetry are included within the scope of this invention. Suchisomers can be obtained in substantially pure form by classicalseparation techniques and by sterically controlled synthesis. For thepurposes of this application, unless expressly noted to the contrary, anamed amino acid shall be construed to include both the (D) or (L)stereoisomers.

A “reversed” or “retro” peptide sequence as disclosed herein refers tothat part of an overall sequence of covalently-bonded amino acidresidues (or analogs or mimetics thereof) wherein the normal carboxyl-toamino direction of peptide bond formation in the amino acid backbone hasbeen reversed such that, reading in the conventional left-to-rightdirection, the amino portion of the peptide bond precedes (rather thanfollows) the carbonyl portion. See, generally, Goodman, M. and Chorev,M. Accounts of Chem. Res. 1979, 12, 423.

The reversed orientation peptides described herein include (a) thosewherein one or more amino-terminal residues are converted to a reversed(“rev”) orientation (thus yielding a second “carboxyl terminus” at theleft-most portion of the molecule), and (b) those wherein one or morecarboxyl-terminal residues are converted to a reversed (“rev”)orientation (yielding a second “amino terminus” at the right-mostportion of the molecule). A peptide (amide) bond cannot be formed at theinterface between a normal orientation residue and a reverse orientationresidue.

Therefore, certain reversed peptide compounds of the invention can beformed by utilizing an appropriate amino acid mimetic moiety to link thetwo adjacent portions of the sequences depicted above utilizing areversed peptide (reversed amide) bond. In case (a) above, a centralresidue of a diketo compound may conveniently be utilized to linkstructures with two amide bonds to achieve a peptidomimetic structure.In case (b) above, a central residue of a diamino compound will likewisebe useful to link structures with two amide bonds to form apeptidomimetic structure.

The reversed direction of bonding in such compounds will generally, inaddition, require inversion of the enantiomeric configuration of thereversed amino acid residues in order to maintain a spatial orientationof side chains that is similar to that of the non-reversed peptide. Theconfiguration of amino acids in the reversed portion of the peptides ispreferably (D), and the configuration of the non-reversed portion ispreferably (L). Opposite or mixed configurations are acceptable whenappropriate to optimize a binding activity.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

(iii) Exemplary Elastin-Based Compositions and Methods

The present invention provides compositions, methods, and devices thatcan be used to promote adhesion and/or migration of endothelial cells.Specifically, the present invention provides compositions and devicescomprising or consisting essentially of tropoelastin, or bioactivefragments thereof. Tropoelastin polypeptides or bioactive fragments foruse in the methods, compositions, and devices of the invention retainone or more of the functional/biological activities of nativetropoelastin. Exemplary biological activities include, but are notlimited to, one or more of the following biological activities: (i)promotes adhesion of endothelial cells in vitro; (ii) promotes adhesionof endothelial cells in vivo; (iii) promotes adhesion of human aorticendothelial cells (HuAEC) in vitro; (iv) promotes adhesion of humanaortic smooth muscle cells (HuAoSMC) in vitro; (v) promotes migration ofendothelial cells in vitro; (vi) promotes migration of human aorticendothelial cells (HuAEC) in vitro; (vii) promotes adhesion of A2058human melanoma cells in vitro; (viii) promotes migration of A2058 humanmelanoma cells in vitro; (ix) promotes adhesion of human microvesselendothelial cells (HMVEC) in vitro.

Polypeptides and Peptide Fragments:

The present invention provides composition and devices comprising orconsisting essentially of tropoelastin polypeptide, or a bioactivefragment thereof. Such compositions and devices can be used in the invivo or in vitro methods of the invention.

In certain embodiments, the compositions or devices comprise atropoelastin polypeptide, or a bioactive fragment thereof. Suchpolypeptides or fragments can include either a wildtype peptide sequenceor a variant sequence, and variant sequences can be readily constructedand tested to ensure that the variant sequence retains one or more ofthe biological activities of the native polypeptide. One of skill in theart can readily make variants comprising an amino acid sequence at least60%, 70%, 75%, 80%, 83%, 85%, 90%, 95%, 98% or 99% identical to aparticular polypeptide, and identify variants that activate elastinsignaling and retain one or more of the biological activities of nativetropoelastin. To further illustrate, the present invention contemplatesvariant polypeptides comprising an amino acid sequence at least 60%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to atropoelastin, or a bioactive fragment thereof (e.g., SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6).

Variants of bioactive fragments of tropoelastin comprise an amino acidsequence at least 60%, 70%, 75%, 80%, 83%, 85%, 90%, 95%, 98%, or 99%identical to the corresponding fragment of native tropoelastin. Suchbioactive fragments retain one or more of the biological activities offull length tropoelastin.

Additionally, certain bioactive fragments are repeated multiple timeswithin the context of the native tropoelastin protein. For example, thehexameric sequence depicted in SEQ ID NO: 5 is repeated multiple timeswithin the context of the native protein. Accordingly, the inventioncontemplates compositions and devices comprising or consistingessentially of one or more repeats of a particular bioactive fragment.For example, the invention contemplates compositions comprising orconsisting essentially of 1, 2, 3, 4, 5, 6, or 7 repeats of thehexameric sequence represented in SEQ ID NO: 5. Additionally, althoughthe C-terminal fragment in not repeated in the context of the nativeprotein, the invention contemplates compositions comprising orconsisting essentially of 1, 2, 3, 4, 5, 6, or 7 repeats of the sequencerepresented in SEQ ID NO: 6. Note that one of skill in the art canreadily make and test compositions comprising multiple repeats of aparticular fragment without undue experimentation.

In addition to the polypeptides and fragments described in detail above,the present invention also pertains to isolated nucleic acids comprisingnucleotide sequences that encode said polypeptides and fragments. Theterm nucleic acid as used herein is intended to include fragments asequivalents, wherein such fragments have substantially the same functionas the full length nucleic acid sequence from which it is derived.Equivalent nucleotide sequences will include sequences that differ byone or more nucleotide substitutions, additions or deletions, such asallelic variants; and will, therefore, include sequences that differfrom the nucleotide sequence of, for example, the wildtype tropoelastin(SEQ ID NO: 1). Equivalent sequences include those that vary from aknown wildtype or variant sequence due to the degeneracy of the geneticcode. Equivalent sequences may also include nucleotide sequences thathybridize under stringent conditions (i.e., equivalent to about 20-27°C. below the melting temperature (T_(m)) of the DNA duplex formed inabout 1M salt) to the nucleotide sequence of an elastin-basedcomposition. Further examples of stringent hybridization conditionsinclude a wash step of 0.2×SSC at 65° C.

In one example, the invention contemplates a composition encoded orencodable by a nucleic acid sequence which hybridizes under stringentconditions, including a wash step of 0.2×SSC at 65° C., to a nucleicacid sequence of SEQ ID NO: 1.

Equivalent nucleotide sequences for use in the methods described hereinalso include sequences which are at least 60% identical to a givenucleotide sequence. In another embodiment, the nucleotide sequence isat least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical tothe nucleotide sequence of a native sequence that encodes anelastin-based composition and retains one or more of the biologicalactivities of the native tropoelastin.

Nucleic acids having a sequence that differs from nucleotide sequenceswhich encode a particular elastin-based composition due to degeneracy inthe genetic code are also within the scope of the invention. Suchnucleic acids encode functionally equivalent peptides but differ insequence from wildtype sequences known in the art due to degeneracy inthe genetic code. For example, a number of amino acids are designated bymore than one triplet. Codons that specify the same amino acid, orsynonyms (for example, CAU and CAC each encode histidine) may result in“silent” mutations which do not affect the amino acid sequence. However,it is expected that DNA sequence polymorphisms that do lead to changesin the amino acid sequences will also exist. One skilled in the art willappreciate that these variations in one or more nucleotides (up to about3-5% of the nucleotides) of the nucleic acids encoding polypeptideshaving one or more of the biological activities of native tropoelastinmay exist among individuals of a given species due to natural allelicvariation.

Peptidomimetics:

In other embodiments, the invention contemplates that the compositioncomprises a peptidomimetic (herein referred to interchangeably as amimetic of an elastin-based composition). Preferable peptidomimeticsretain one or more of the biological activities of native tropoelastin.Peptidomimetics are compounds based on, or derived from, peptides andproteins. The peptidomimetics of the present invention can be obtainedby structural modification of the amino acid sequence of a knownelastin-based composition using unnatural amino acids, conformationalrestraints, isosteric replacement, and the like. The subjectpeptidomimetics constitute the continuum of structural space betweenpeptides and non-peptide synthetic structures.

Exemplary peptidomimetics can have such attributes as beingnon-hydrolyzable (e.g., increased stability against proteases or otherphysiological conditions which degrade the corresponding peptide),having increased specificity and/or potency, and having increased cellpermeability for intracellular localization. For illustrative purposes,peptide analogs of the present invention can be generated using, forexample, benzodiazepines (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gama lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988, p123), C-7 mimics (Huffman et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988, p. 105), keto-methylene pseudopeptides(Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. inPeptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), β-turndipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1:1231), β-aminoalcohols (Gordonet al. (1985) Biochem Biophys Res Commun 126:419; and Dann et al. (1986)Biochem Biophys Res Commun 134:71), diaminoketones (Natarajan et al.(1984) Biochem Biophys Res Commun 124:141), and methyleneamino-modified(Roark et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988, p 134). Also, see generally,Session III: Analytic and synthetic methods, in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988)

In addition to a variety of sidechain replacements which can be carriedout to generate the subject peptidomimetics, the present inventionspecifically contemplates the use of conformationally restrained mimicsof peptide secondary structure. Numerous surrogates have been developedfor the amide bond of peptides. Frequently exploited surrogates for theamide bond include the following groups (i) trans-olefins, (ii)fluoroalkene, (iii) methyleneamino, (iv) phosphonamides, and (v)sulfonamides.

Examples of Surrogates

Additionally, peptidomimetics based on more substantial modifications ofthe backbone of a peptide can be used. Peptidomimetics which fall inthis category include (i) retro-inverso analogs, and (ii) N-alkylglycine analogs (so-called peptoids).

Examples of Analogs

Furthermore, the methods of combinatorial chemistry are being brought tobear, e.g., PCT publication WO 99/48897, on the development of newpeptidomimetics. For example, one embodiment of a so-called “peptidemorphing” strategy focuses on the random generation of a library ofpeptide analogs that comprise a wide range of peptide bond substitutes.

In an exemplary embodiment, the peptidomimetic can be derived as aretro-inverso analog of the peptide. Retro-inverso analogs can be madeaccording to the methods known in the art, such as that described by theSisto et al. U.S. Pat. No. 4,522,752. As a general guide, sites whichare most susceptible to proteolysis are typically altered, with lesssusceptible amide linkages being optional for mimetic switching. Thefinal product, or intermediates thereof, can be purified by HPLC.

In another illustrative embodiment, the peptidomimetic can be derived asa retro-enantio analog of the peptide, such as the exemplaryretro-enantio peptide analog derived for the illustrative Val Gly ValAla Pro Gly peptide (SEQ ID No. 5):

-   -   NH2-(d)Gly-(d)Pro-(d)Ala-(d)Val-(d)Gly-(d)Val-COOH (SEQ ID No.        7)        Retro-enantio analogs such as this can be synthesized using        commercially available D-amino acids (or analogs thereof) and        standard solid- or solution-phase peptide-synthesis techniques.        For example, in a preferred solid-phase synthesis method, a        suitably amino-protected (t-butyloxycarbonyl, Boc) residue (or        analog thereof) is covalently bound to a solid support such as        chloromethyl resin. The resin is washed with dichloromethane        (DCM), and the BOC protecting group removed by treatment with        TFA in DCM. The resin is washed and neutralized, and the next        Boc-protected D-amino acid is introduced by coupling with        diisopropylcarbodiimide. The resin is again washed, and the        cycle repeated for each of the remaining amino acids in turn.        When synthesis of the protected retro-enantio peptide is        complete, the protecting groups are removed and the peptide        cleaved from the solid support by treatment with hydrofluoric        acid/anisole/dimethyl sulfide/thioanisole. The final product is        purified by HPLC to yield the pure retro-enantio analog.

In still another illustrative embodiment, trans-olefin derivatives canbe made for any of the subject polypeptides. A trans olefin analog canbe synthesized according to the method of Y. K. Shue et al. (1987)Tetrahedron Letters 28:3225 and also according to other methods known inthe art. It will be appreciated that variations in the cited procedure,or other procedures available, may be necessary according to the natureof the reagent used.

It is further possible to couple the pseudodipeptides synthesized by theabove method to other pseudodipeptides, to make peptide analogs withseveral olefinic functionalities in place of amide functionalities.

Still another classes of peptidomimetic derivatives include phosphonatederivatives. The synthesis of such phosphonate derivatives can beadapted from known synthesis schemes. See, for example, Loots et al. inPeptides: Chemistry and Biology, (Escom Science Publishers, Leiden,1988, p. 118); Petrillo at al. in Peptides: Structure and Function(Proceedings of the 9th American Peptide Symposium, Pierce Chemical Co.Rockland, Ill., 1985).

Many other peptidomimetic structures are known in the art and can bereadily adapted for use in designing elastin-based compositionpeptidomimetics. To illustrate, the peptidomimetic may incorporate the1-azabicyclo[4.3.0]nonane surrogate (see Kim et al. (1997) J. Org Chem.62:2847), or an N-acyl piperazic acid (see Xi at al. (1998) J. Am. Chem.Soc. 120:80), or a 2-substituted piperazine moiety as a constrainedamino acid analogue (see Williams et al. (1996) J. Med. Chem.39:1345-1348). In still other embodiments, certain amino acid residuescan be replaced with aryl and bi-aryl moieties, e.g., monocyclic orbicyclic aromatic or heteroaromatic nucleus, or a biaromatic, aromatic,heteroaromatic, or biheteroaromatic nucleus.

The subject peptidomimetics can be optimized by, e.g., combinatorialsynthesis techniques combined with high throughput screening techniques,and furthermore can be tested to ensure that the peptidomimetic retainsone or more of the biological activities of native tropoelastin.

The invention contemplates methods, compositions, and devices forpromoting adhesion and/or migration of endothelial cells. The inventioncontemplates the use of a single elastin composition (e.g., atropoelastin polypeptide; a single bioactive fragment; a singlepeptidomimetic), as well as the use of more than one elastin compositionused concurrently or consecutively. Accordingly, the inventioncontemplates embodiments in which a tropoelastin polypeptide and one ormore tropoelastin fragments or peptidomimetics are used concurrently orconsecutively. The invention contemplates embodiments in which two ormore tropoelastin fragments or peptidomimetics are used.

The present invention provides methods, compositions, and devices topromote adhesion and/or migration of endothelial cells. Tropoelastin, orone or more bioactive fragments thereof, can be used to promoteendothelial adhesion and/or migration. In certain embodiments,tropoelastin, or a bioactive fragment thereof, is covalently appended toa device, and the tropoelastin, or a bioactive fragment thereof,promotes adhesion of endothelial cells to the metal device. Theinvention contemplates methods of promoting endothelial cell adhesionusing any of the following: tropoelastin; a bioactive fragment thereof;a peptidomimetic; more than one bioactive fragment thereof; more thanone peptidomimetic; or a combination of full length tropoelastin and oneor more bioactive fragments or peptidomimetics thereof.

The present invention provides methods, compositions, and devices topromote adhesion and/or migration of endothelial cells. Exemplaryendothelial cells include, but are not limited to, endothelial stemcells. The methods, compositions, and devices of the invention can beused in vitro or in vivo. Endothelialization of devices can occur invivo following device placement. In vivo endothelialization occurs asendogenous endothelial cells adhere to the device. Endothelialization ofdevices can also occur in vitro prior to device placement. In otherwords, a device can be pre-seeded with endothelial cells, for exampleendothelial cells derived from the patient, prior to placement. Furtherendothelialization may occur following device placement.

The effect of elastin on endothelial cell migration and proliferationoffers unique therapeutic advantages to prevent some of thecomplications that are associated with the placement of intravasculardevices, such as coronary stents. Recently deployed coronary stentscarry with them increased rates of in situ thrombosis, which rates ofthrombosis decrease dramatically once the metal struts of the stent haveendothelialized. This necessitates the use of multiple antiplateletagents, such as aspirin and clopidogrel, until such endothelializationhas occurred. While usually tolerated well, such antiplatelet use cancause treatment dilemmas when surgical procedures are required or whenthe patients have disease such as atrial fibrillation that require othermethods of anticoagulation, such as inhibition of vitamin K-dependentcoagulation factors. Materials that promote more rapid intravascularendothelialization of intravascular devices would not only reduce insitu rates of thrombosis but also could potentially decrease thenecessary time for dual anti-platelet therapy.

Accordingly, the present invention provides methods for reducingthrombosis. The present invention further provides methods for reducingthe necessary treatment time for anti-platelet therapy.

An additional complication of coronary stent placement is the subsequentrisk of smooth muscle cell hyperproliferation and in stent restenosis,especially in high risk individuals such as those with diabetes.Drug-eluting stent platforms have been developed in an attempt to combatthis restenosis. Through the elution of cytoxic agents, drug-elutingstents have largely overcome the problem of restenosis and have extendedthe reach of interventional cardiology to include high risk lesions thatpreviously would have solidly been within the domain of cardiothoracicsurgery. However, drug-eluting stents have also perhaps increased thetime frame during which in situ thrombosis may occur, with cases of instent thrombosis occurring sometimes >1 year after drug-eluting stent(DES) placement. This likely results from delayed endothelializationthat occurs with prolonged elution of the cytotoxic agent.

The use of tropoelastin, or bioactive fragments thereof, may provide anopportunity to overcome both of these limitations. Here we demonstratethat elastin promotes endothelial migration. Such enhanced migration mayresult in more rapid device endothelialization by migration/invasion bythe surrounding endothelium. Additionally, we demonstrate that stainlesssteel surfaces with covalently attached elastin have markedly increasedadherence of endothelial cells and endothelial progenitor cells thansimilar bare metal surfaces. In addition, the adherent endothelial cellshave a spread cytoplasm as they might after adhesion to an intactinternal elastic lamina. Given the ability of tropoelastin, andbioactive fragments thereof, to promote adhesion and/or migration ofendothelial cells, these polypeptides can be used in methods and devicesto decrease thrombosis and restenosis. The polypeptides can be usedalone or can be used in combination with other devices or therapies.

In certain embodiments, the invention provides methods for promotingendothelial cell adhesion using a combination of tropoelastin (or afragment) with one or more other agents that promote adhesion. Forexample, tropoelastin (or a fragment) can be used concurrently orconsecutively with an anti-CD34 antibody. In certain embodiments, theinvention provides methods and devices using both an elastin-basedcomposition and anti-CD34 antibody.

Previously we have described the effect of elastin on smooth musclemigration and differentiation and have demonstrated the ability of anelastin sheath to reduce in stent intimal hyperplasia. Stainless steeldevices coated with elastin may not only endothelialize rapidly buy mayhave the added benefit of reduced subsequent intimal hyperplasia.

The foregoing description of intravascular thrombosis following stentplacement provides a specific example illustrating a general problemassociated with the placement of any device into the body or into avessel lumen. Thrombosis is a problem associated with placement ofstents, catheters, wires, shunts, and other intraluminal devices,regardless of whether the devices are placed intravascularly or intoanother type of vessel. Furthermore, thrombosis is a problem associatedwith the placement of larger devices includingcardioverter-defibrillators, pacemakers, chest tubes, ventricular-assistdevices, ventilators, patent foramen ovale closure devices, and thelike. The risk of thrombosis often necessitates antiplatelet oranticoagulant management, and increases the overall risk of theseprocedures. On a whole, the risks associated with thrombosis greatlyinfluence interventional treatment of a wide range of diseases andconditions including, but not limited to cardiovascular conditions.

In certain embodiments, the amount of tropoelastin, or a bioactivefragment thereof, effective to promote adhesion of endothelial cells isan amount sufficient to promote and maintain adhesion of endothelialcells under physiologically relevant conditions of flow rate andpressure. Such physiologically relevant conditions are the flow rate andpressure conditions encountered in an artery or vein, or fluid flow rateand pressure conditions encountered within another body vessel. The useof amounts of tropoelastin, or a bioactive fragment thereof, effectiveto maintain adhesion under physiological conditions of arterial and/orvenous blood flow and pressure, or fluid flow and pressure conditionsencountered within another body vessel, is an important step in theeffective in vivo use of tropoelastin compositions and devices appendedwith tropoelastin compositions.

In certain embodiments, the amount of tropoelastin or a bioactivefragment used to promote adhesion and/or migration is an amounteffective to promote and maintain adhesion of endothelial cells underphysiologically relevant conditions. For example, the amount oftropoelastin or a bioactive fragment covalently attached to a devicecomprises an amount effective to promote and maintain adhesion ofendothelial cells under physiologically relevant conditions. Exemplaryeffective amounts are at least about 100 ng/square centimeter, at leastabout 150 ng/square centimeter, or at least about 200 ng/squarecentimeter of device surface. Further exemplary effective amounts are atleast about 250 ng/square centimeter, at least about 275 ng/squarecentimeter, or at least about 300 ng/square centimeter. Still additionalexemplary effective amounts are at least about 310 ng/square centimeter,at least about 325 ng/square centimeter, at least about 350 ng/squarecentimeter, at least about 375 ng/square centimeter, or at least about400 ng/square centimeter. Still additional exemplary effective amountsare at least about 450 ng/square centimeter, at least about 500ng/square centimeter, at least about 550 ng/square centimeter, at leastabout 600 ng/square centimeter, at least about 625 ng/square centimeter,or at least about 650 ng/square centimeter, or at least about 700ng/square centimeter. The invention recognizes and contemplates that theamount effective to promote and maintain adhesion under physiologicallyrelevant conditions may vary depending on the particular vessel intowhich the device is placed, as well as the underlying disease state orphysiology of the patient. Furthermore, the effective amount may varydepending on the device material, as some materials are morethrombogenic than other materials. For example, metal is particularlythrombogenic, and the risk of thrombosis following placement of a metaldevice is greater than the risk of thrombosis following placement ofdevices made from other materials. One of skill in the art can determinethe optimal conditions for treating a particular patient.

Exemplary devices may be made from or coated with any of a number ofmaterials. As detailed throughout, the invention contemplates attachingtropoelastin or bioactive fragments thereof to virtually any devicethat, in use, is placed into a vessel in a human or animal body.Exemplary materials are biocompatible materials typically used in themanufacture of medical devices. By way of examples, devices may be madefrom or coated with metal, plastic, silicone, dacron, PTFE, orderivatives or alloys thereof. The subject polypeptides can becovalently attached to or crosslinked to the device surface. Methods ofcovalently attaching polypeptides to solid surfaces are well known inthe art. One example is provided herein. However, other methods ofattaching polypeptides to surfaces known in the art are similarlycontemplated and can be readily used. One of skill in the art can selectamongst various attachment chemistries and methods depending on theconcentration of protein to be attached, and the nature of the devicesurface to which attachment is desired.

(iv) Method of Screening

This application describes methods and compositions for promotingadhesion and migration of endothelial cells and endothelial progenitorcells using compositions comprising tropoelastin, or bioactive fragmentsthereof. With the importance of providing effective devices, methods,and compositions for preventing and/or decreasing restenosis andthrombosis, the present invention recognizes the utility in screening toidentify tropoelastin variant polypeptides, bioactive fragments oftropoelastin, as well as peptidomimetics that might be useful inpromoting adhesion and/or migration of endothelial cells. Exemplaryfragments, variants, and peptidomimetics identified and/or characterizedby the methods of the present invention retain one or more of thefollowing biological activities of native tropoelastin: (i) promotesadhesion of endothelial cells in vitro; (ii) promotes adhesion ofendothelial cells in vivo; (iii) promotes adhesion of human aorticendothelial cells (HuAEC) in vitro; (iv) promotes adhesion of humanaortic smooth muscle cells (HuAoSMC) in vitro; (v) promotes migration ofendothelial cells in vitro; (vi) promotes migration of human aorticendothelial cells (HuAEC) in vitro; (vii) promotes adhesion of A2058human melanoma cells in vitro; (viii) promotes migration of A2058 humanmelanoma cells in vitro; (ix) promotes adhesion of human microvesselendothelial cells (HMVEC) in vitro. Variants, fragments, andpeptidomimetics identified by the screening methods of the invention canthen be further analyzed to determine whether they are useful in vivofor treating or preventing restenosis and/or thrombosis.

The screening methods contemplated include screening single candidatepolypeptides, as well as libraries of polypeptides. Furthermore, thescreening methods can be conducted as high throughput assays.

In the context of the present invention, a cell based screen can beconducted in endothelial cells or endothelial cell lines in culture.Exemplary endothelial cells or cell lines can be derived from anyspecies including, but not limited to, humans, mice, rats, rabbits,cows, pigs, non-human primates, and the like. Furthermore, exemplaryendothelial cells include endothelial progenitor cells or transformedendothelial cell lines. By way of non-limiting example, the screen canbe conducted in cultures of human aortic endothelial cells (HuAECs) orhuman microvessel endothelial cells (HMVECs).

Regardless of the particular cell type used, cultures of the cells canbe contacted with one or more candidate polypeptides and assayed forcell adhesion and/or cell migration. As a control, the effect of thecandidate polypeptide can be compared to behavior of the cells in theabsence of polypeptide or in the presence of a carrier such as BSA.Using this method, potentially useful polypeptide variants, fragments,or peptidomimetics can be identified and/or characterized. Specifically,polypeptides that promote adhesion and/or migration of endothelial cellsin comparison to the behavior of the cells in the absence of polypeptideor in the presence of carrier only, are polypeptides that may be usefulin the methods of the present invention. Such polypeptides can befurther analyzed using additional in vitro or in vivo assays.

Regardless of the methodology used to identify and/or characterize atropoelastin polypeptide variant, fragment, or peptidomimetic,polypeptides identified as promoting endothelial adhesion and/ormigration in vitro have a variety of in vitro or in vivo uses.Accordingly, the invention further contemplates the use of a polypeptideidentified by the screening methods of the invention. Identifiedelastin-based compositions may be used alone or in combination withother agents, or may be formulated in a pharmaceutically acceptablecarrier.

The invention also contemplates various methods for testing the efficacyof various devices to which tropoelastin or peptide fragments have beenattached. For example, devices made of various materials (e.g., plastic,metal, PTFE, silicone) can be coated with an effective amount oftropoelastin or bioactive fragment via crosslinking or via a covalentlinkage. Coated devices can be inserted into the vessel of an animalmodel. For example, pigs are often used as an animal model forcardiovascular indications. Coated devices can be placed into an arteryor vein of a pig. The thrombogenic effect of the implanted device can becompared to the thrombogenic effect of a bare metal device placed intothe artery or vein of a control animal. Given that metal is typicallythe most thrombogenic of device materials, the use of a bare metalcontrol represents the maximal level of device thrombosis.

Imaging can be used to view the implanted device. At some pointfollowing the experiment, the device can be removed and imaged to assessthe relative endothelialization of the various coated devices incomparison to the bare metal device.

Similar experiments can be conducted using other vessels and otheranimal models. Furthermore, experiments can be conducted using injuredor diseased animals. Given that medical devices are implanted in sick orinjured patients, the use of sick animals may provide a more accuratemodel of the thrombogenic process.

(v) Methods of Administration of Nucleic Acids, Proteins, ChemicalCompounds and Pharmaceutical Compositions of Agents

The invention further contemplates pharmaceutical compositions includingtropoelastin polypeptide, and bioactive fragments thereof. Thepharmaceutical compositions comprise a tropoelastin polypeptide, andbioactive fragments thereof formulated, in one embodiment, according toconventional pharmaceutical compounding techniques. See, for example,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa.). Pharmaceutical formulations of the invention cancontain the active agent or a pharmaceutically acceptable salt of theactive agent. These compositions can include, in addition to an activeagent, a pharmaceutically acceptable excipient, carrier, buffer,stabilizer or other material well known in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive agent. Preferable pharmaceutical compositions are non-pyrogenic.Additional preferred pharmaceutical compositions are non-antigenic. Thecarrier may take a wide variety of forms depending on the route ofadministration, e.g., intravenous, intravascular, oral, intrathecal,epineural or parenteral, transdermal, pulmonary, etc. Additionally, thecarrier is appropriate for covalent attachment of the tropoelastinpolypeptide, or bioactive fragment thereof, to a metal device.

Illustrative examples of suitable carriers are water, saline, dextrosesolutions, fructose solutions, ethanol, or oils of animal, vegetative orsynthetic origin. The carrier may also contain other ingredients, forexample, preservatives, suspending agents, solubilizing agents, buffersand the like.

Pharmaceutical compositions according to the invention include devices.Such devices are covalently conjugated with a composition comprisingtropoelastin, or a bioactive fragment thereof. Tropoelastin compositionscan also be cross-linked to a device surface. Coated devices areappropriate for administration (e.g., insertion, implantation, delivery)into a human or animal body. Coated devices have optionally beenapproved by the FDA for one or more indications.

Exemplary devices are metal devices of virtually any shape or sizeappropriate for the particular indication. Exemplary metals can bechosen by one of skill in the art but include, without limitationstainless steel, surgical steel, titanium, gold, silver, platinum,nitinol, aluminum, nickel, and other metals and metal alloys appropriatefor implantation into a human or animal body. Further exemplary devicesare made of or coated with plastic, silicone, Dacron, PTFE, orderivatives or combinations thereof.

Exemplary devices include, without limitation, stents, catheters, wires,and other intraluminal devices. Further exemplary devices includeshunts, chest tubes, patent foramen ovale closure devices,cardioverter-defibrillators, pacemakers, endovascular grafts, mechanicalhearts, mechanical heart valves, ventricular-assist devices, andventilators. Such devices can be delivered intravenously,intravascularly, intraarterially, orally, rectally, surgically,urethrally, or by other methods known in the art for appropriatelydelivering a particular device.

One of skill in the art will readily recognize that thrombosis andrestenosis are issues faced not only in association with the treatmentof cardiovascular diseases, but also in other occlusive body vesseldiseases and disorders. Furthermore, thrombosis is a potential issueassociated with the implantation of any medical device regardless of theparticular indication for which the medical device constitutes atreatment. Accordingly, the invention contemplates methods and devicesfor use in a variety of body vessels and in association with a varietyof disease indications. By way of example, exemplary body vesselsinclude, but are not limited to, artery, vein, common bile duct,pancreatic duct, kidney duct, esophagus, trachea, urethra, bladder,uterus, ovarian duct, Fallopian tube, vas deferens, prostatic duct, orlymphatic duct. Further specific devices appropriate fornon-cardiovascular indications include, but are not limited to,gallbladder stents, endotracheal stents, bladder drainage catheters,cerebral-peritoneal shunt catheters, dialysis catheters, grafts orcatheters for peritoneal dialysis, and the like. In short, the inventionis based on the recognition and appreciation that virtually everymedical intervention that involves the placing of a device, temporarilyor permanently, into a human or animal body creates a risk ofthrombosis. Accordingly, the present invention provides methods andcompositions that can be used as part of virtually any such medicalintervention to reduce the risk of thrombosis.

The invention also provides articles of manufacture includingpharmaceutical compositions of the invention, devices, and related kits.The invention encompasses any type of article including a pharmaceuticalcomposition of the invention, but the article of manufacture istypically a container, preferably bearing a label identifying thecomposition contained therein, as well as instructions for use of thecomposition and/or device.

In another embodiment of any of the foregoing, the inventioncontemplates that a device coated with tropoelastin, or bioactivefragment thereof, can further be pre-coated (coated prior toimplantation into a human or non-human patient) with endothelial cells.In such embodiments, endothelial cells or endothelial progenitor cells(e.g., either harvested from the same patient or from a related orunrelated donor) would be adhered to the tropoelastin-coated devices exvivo. These devices would then be implanted into the patient. Withoutbeing bound by theory, once implanted into the body, the patient's owncells may additionally adhere to the device. However, regardless ofwhether further endothelial adhesion occurred in vivo, the device wouldalready be coated with endothelial cells or endothelial progenitorcells, thereby decreasing or preventing restenosis or thrombosis.

One of skill in the art will recognize that ex vivo seeding of thedevice with endothelial cells derived from a source other than the samepatient could provoke an immune response upon implantation. Accordingly,in one embodiment, the invention contemplates co-administeringanti-rejection drugs.

The invention includes a method for prophylaxis or treatment ofrestenosis or thrombosis associated with the treatment of anycardiovascular or other disorder that requires implantation of a medicaldevice. Exemplary disorders include cardiovascular diseases, obstructivevascular diseases, and the like. By way of example, the inventioncontemplates methods for the prophylaxis or treatment of restenosis orthrombosis following treatment for atherosclerosis, restenosis, vascularbypass graft stenosis, transplant arteriopathy, aneurysm, anddissection. Furthermore, the invention contemplates methods for theprophylaxis or treatment of restenosis or thrombosis following treatmentof vessels selected from common bile duct, pancreatic duct, esophagus,trachea, urethra, bladder, uterus, ovarian duct, Fallopian tube, vasdeferens, prostatic duct, tear duct, and lymphatic duct. Additionally,the invention recognizes that patients often require device placement aspart of the treatment for any of a wide range of diseases andconditions. By way of example, patients suffering from virtually anydisease, injury, or condition may need to be placed on a ventilator. Inthe cases of some patients, placement on a ventilator may be a long termpart of treatment. For example, patients with ALS or severe spinal cordinjuries, especially those occurring at a high level of the spinal cord,may require long term maintenance on a ventilator. By way of furtherexample, patients on dialysis may require long term use of a peritonealcatheter. The invention contemplates methods, compositions, and devicesfor decreasing or preventing thrombosis occurring following deviceplacement associated with the treatment of any disease, condition, orinjury.

Regardless of whether the compositions of the invention are used invitro or in vivo, and regardless of whether they are covalently attachedto a device, the invention contemplates that the compositions of theinvention can be conveniently formulated for administration with abiologically acceptable medium, such as water, buffered saline, polyol(for example, glycerol, propylene glycol, liquid polyethylene glycol andthe like) or suitable mixtures thereof.

Optimal concentrations of the active ingredient(s) in the chosen mediumcan be determined empirically, according to procedures well known tomedicinal chemists. As used herein, “biologically acceptable medium”includes solvents, dispersion media, and the like which may beappropriate for the desired route of administration of the one or moreagents. The use of media for pharmaceutically active substances is knownin the art. Except insofar as a conventional media or agent isincompatible with the activity of a particular agent or combination ofagents, its use in the pharmaceutical preparation of the invention iscontemplated. Suitable vehicles and their formulation inclusive of otherproteins are described, for example, in the book Remington'sPharmaceutical Sciences (Remington's Pharmaceutical Sciences. MackPublishing Company, Easton, Pa., USA 1985). These vehicles includeinjectable “deposit formulations”.

Methods of introduction may also be provided by delivery via abiocompatible, device. Biocompatible devices suitable for delivery ofthe subject agents include intraluminal devices such as stents, wires,catheters, sheaths, and the like. However, administration is not limitedto delivery via a biocompatible device. As detailed herein, the presentinvention contemplates any of number of routes of administration andmethods of delivery.

The effective amount or dosage level will depend upon a variety offactors including the activity of the particular one or more agentsemployed, the route of administration, the time of administration, therate of excretion of the particular agents being employed, the durationof the treatment, other drugs, compounds and/or materials used incombination with the particular agents employed, the age, sex, weight,condition, general health and prior medical history of the animal, andlike factors well known in the medical arts.

Agents can be administered alone, or can be administered as apharmaceutical formulation (composition). Said agents may be formulatedfor administration in any convenient way for use in human or veterinarymedicine. In certain embodiments, the agents included in thepharmaceutical preparation may be active themselves, or may be aprodrug, e.g., capable of being converted to an active compound in aphysiological setting.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Tropoelastin Promotes Adhesion of Endothelial Cells

FIG. 1 shows the effect of fibronectin and tropoelastin on the adhesionof CHO cells, human embryonic kidney cells (HEK 293), human aorticsmooth muscle cells (HuAoSMC), and human aortic endothelial cells(HuAEC). Cells were plated on culture plates coated with either 0.1% BSA(as a control), 10 ug/ml fibronectin (FN), or 10 ug/ml recombinanttropoelastin, and assayed for adherence to the coated substrate.

All four of the cell types examined adhered to fibronectin. In contrast,the HuAECs and, to a less extent, the HuAoSMCs, adhered to tropoelastin.However, neither CHO cells nor HEK 293 cells adhered to tropoelastin.These results demonstrated that endothelial cells and smooth musclecells adhere to tropoelastin. However, unlike the binding of cells tofibronectin, there is some cell-type specificity to the binding of cellsto tropoelastin. For example, FIG. 1 shows that CHO cells and HEK 293cells, which do adhere to fibronectin, do not adhere to tropoelastin.Note that the full length, recombinant tropoelastin used throughoutthese experiments is represented in SEQ ID NO: 4. Specifically, theseexperiments were conducted with recombinant human tropoelastin lacking anative signal sequence. Furthermore the protein was engineered to have a10× His tag on the N-terminus followed by a Factor Xa cleavage site. Thetotal length of the human tropoelastin protein used in these experimentswas 706 amino acid residues.

The results shown in FIG. 1 are depicted graphically in FIG. 2. FIG. 2shows that tropoelastin promotes adhesion of HuAECs. This cell adhesion,as measured by the number of cells bound/well, is comparable to thatobserved with fibronectin or collagen.

To determine whether individual combinations of integrins were involvedin cell adhesion to wells coated with fibronectin, collagen, ortropoelastin, endothelial cells were incubated with either 25 μg/mlimmunoglobulin G (IgG, control) or with 25 μg/ml anti-α_(v)β₃ oranti-α₂β₁ blocking antibodies prior to exposing the cells to the coatedsurfaces. Preincubation with the anti-α_(v)β₃ or anti-α₂β₁ antibodiesdid not significantly decrease endothelial cell adhesion to surfacescoated with fibronectin or collagen. However, endothelial adhesion towells coated with tropoelastin was blocked by more than 50% byincubation of the cells with anti-α_(v)β₃ blocking antibodies. Thissuggested that binding of tropoelastin to the integrin α_(v)β₃ is animportant contributor to endothelial adhesion to tropoelastin-coatedsurfaces.

Example 2 Tropoelastin, and Bioactive Fragments Thereof, PromoteMigration of Endothelial Cells

FIG. 3 graphically summarizes resulting demonstrating that tropoelastinpromoted migration of endothelial cells. Migration of HuAECs to BSA,fibronectin, and recombinant tropoelastin was measured. Recombinanttropoelastin promoted endothelial cell migration. Furthermore,tropoelastin promoted cell migration at a level comparable to that offibronectin.

We additionally evaluated the ability of two bioactive fragments oftropoelastin to promote endothelial cell migration, and these resultsare also summarized in FIG. 3. We assayed the hexamer VGVAPG (SEQ ID No.5). This hexameric sequence is repeated multiple times within the fulllength tropoelastin protein. Furthermore, a single repeat of thehexamer, as well as multiple repeats of the hexamer, have been shown toretain at least some of the biological activities of the full lengthtropoelastin protein. We additionally assayed a 17 residue C-terminalfragment of the tropoelastin protein. This 17 amino acid residue isdepicted in SEQ ID NO: 6 (IFPGGACLGKACGRKRK).

As summarized in FIG. 3, both the hexameric sequence and the C-terminal17 amino acid residue fragment promoted migration of endothelial cells.The fragments functioned equally well in this assay. Furthermore, thefragments promoted endothelial cell migration at least as well as, andpossibly better than, the full length tropoelastin protein.

FIG. 3 Tropoelastin, and Bioactive Fragments Thereof, Promote Adhesionand Migration of A2058 Human Melanoma Cells

FIG. 4 shows the effect of fibronectin, tropoelastin, and a bioactivefragment of tropoelastin on the adhesion of A2058 human melanoma cells.A2058 cells were assayed for adherence to substrates coated with BSA(control), fibronectin, tropoelastin, or a bioactive fragment oftropoelastin corresponding to a single repeat of the hexameric sequenceVGVAPG (SEQ ID No. 5). In comparison to adherence to BSA, A2058 cellsadhered to substrates coated with fibronectin, tropoelastin, or abioactive fragment of tropoelastin. Specifically, adhesion totropoelastin was dose dependent, and cells adhered to 50 μg/ml oftropoelastin similarly to 10 μg/ml of fibronectin. In addition, 10 μM ofa single repeat of the hexameric sequence VGVAPG (SEQ ID No. 5) (e.g., abioactive fragment of tropoelastin) promoted adhesion of A2058 cells, incomparison to BSA, and at a level similar to 10 μg/ml of full lengthtropoelastin.

FIG. 5 shows the effect of fibronectin, tropoelastin, and two differentbioactive fragments of tropoelastin on the migration of A2058 humanmelanoma cells. A2058 cells migrated in response to fibronectin,tropoelastin, a single repeat of the hexameric sequence VGVAPG (SEQ IDNo. 5) (e.g., a bioactive fragment of tropoelastin), and a C-terminalfragment of tropoelastin (the C-terminal fragment represented in SEQ IDNO: 6). Full length tropoelastin, a single repeat of VGVAPG (SEQ ID No.5), and the C-terminal fragment represented in SEQ ID NO: 6 all promotedmigration of A2058 cell at comparable levels.

Example 4 Endothelial Cells Adhere to Coated Stainless Steel Devices

The present invention is based on the finding that tropoelastin, andbioactive fragments thereof, promote adhesion and migration ofendothelial cells. This finding allows the design of methods andcompositions to promote adhesion of endothelial cells to devices thatare or can be implanted into human or non-human animals. As outlined indetail throughout the application, the coating of devices withendothelial cells (e.g., either prior to placement in the body or invivo) helps prevent restenosis and thrombosis.

Given that an important aspect of the present invention is theprevention of restenosis and thrombosis, we conducted experiments toaddress whether endothelial cells would adhere to metal devices that hadbeen coated with tropoelastin. Briefly, tropoelastin was covalentlyconjugated to a stainless steel disk approximately 6 mm in diameter.Tropoelastin was linked to the disk surface using an intermediatepolysaccharide molecule. An exemplary process for covalently linkingtropoelastin to the disk surface, and the process used here, is used inthe manufacture of Genous Bio-Engineered R Stents. These disks providedample surface area for microscopic imaging. Disks were also similarlycoated with anti-CD34 antibody and with polysaccharide vehicle alone.Stents coated with anti-CD34 antibody using a similar process (GenousBio-Engineered R Stents) are currently in human clinical trials and havepromise to promote capture of circulating endothelial cells andendothelial progenitor cells and to accelerate endothelialization.

Human microvessel endothelial cells (HMVEC) were cultured with thesecoated disks, and adhesion of the cells to the coated disks was assayed.The results of these experiments are summarized in FIG. 6. Tropoelastinpromoted adhesion of human endothelial cells to a metal device.

Example 5 Endothelial Cells Adhere to and Spread Across the Surface ofCoated Stainless Steel Devices

To prevent or treat thrombosis and restenosis, the present inventionprovides methods and devices for promoting adhesion and/or migration ofendothelial cells. In one embodiment, endothelial cells bind to animplantable medical device, thereby decreasing or preventing thrombosisor restenosis. The invention contemplates that the device could becoated with endothelial cells (e.g., either a patient's own endothelialcells or endothelial cells derived from another person or from ananimal). Alternatively, the invention contemplates that, followingimplantation of the device into a patient, the patient's own endothelialcells (e.g., endothelial cells or endothelial progenitor cells) wouldadhere to the implanted device. Without being bound by theory, the cellscould coat the device, in whole or in part, thereby helping to preventor decrease thrombosis and/or restenosis.

We conducted experiments to assess whether tropoelastin promoted theadhesion of endothelial progenitor cells. Tropoelastin was covalentlyconjugated to a stainless steel disk approximately 6 mm in diameter. Asa control, anti-CD34 antibody (known to promote endothelial celladhesion) was covalently conjugated to similar stainless steel disks.Endothelial progenitor cells were cultured with these coated disks, andadhesion of the cells to the coated disks was assayed. The results ofthese experiments are summarized in FIG. 7. Tropoelastin promotedadhesion of human endothelial cells to the metal device.

We also examined the appearance of the adherent endothelial cells. Weobserved that tropoelastin not only promoted adhesion of the cells tothe metal device, but furthermore, promoted spreading of the adherentcells across the surface of the device. This cell spreading was uniqueto the device coated with tropoelastin and was not observed with thedevice coated with an anti-CD34 antibody.

Cell spreading was examined by permeabilizing the endothelial cells thathad adhered to the coated metal surfaces with Triton X-100 and stainingwith phalloidin to highlight the actin cytoskeleton and DAPI to markcell nuclei. As noted above, metal disks coated with either anti-CD34antibody or with tropoelastin resulted in a significant increase in celladhesion compared to polysaccharide-coated control disks or to baremetal disks. Endothelial cells adherent to tropoelastin-coated disks hadwell spread cytoplasm and an organized actin cytoskeleton. Thus,stainless steel surfaces coated with either tropoelastin or anti-CD34antibody would likely endothelialize more rapidly than would similarbare metal surfaces when intravascularly placed.

The invention contemplates use of metal devices coated withtropoelastin, or a bioactive fragment thereof, to promote adhesion ofendothelial cells to the metal device. The invention also contemplatesthe use of metal devices coated with both tropoelastin, or a bioactivefragment thereof, and with anti-CD34 antibody. Given the ability of bothagents to promote adhesion of endothelial cells, the use of co-coateddevices may be advantageous in some instances.

The foregoing experiments used the following methods:

Cell Adhesion Assays

96-well plates were coated in duplicate overnight at 4° C. with 10 μg/mlcollagen, fibronectin, tropoelastin, or 0.1% bovine serum albumin (BSA).Cells were harvested with 0.25% trypsin/1 mM EDTA, washed three times inadhesion buffer (0.5% BSA in basal medium), suspended at a workingdensity of 5×10/ml in adhesion buffer, and then allowed to recover for 1h at 37° C. (5% CO₂). For integrin-blocking assays, 25 μg/mlanti-integrin antibodies (Chemicon, Temecula, Calif.) or control IgG(Jackson ImmunoResearch Laboratories, Inc, West Grove, Pa.) were addedto the cell suspension during the recovery period. During the cellrecovery, the coated 96-well plates were washed twice in dPBS andblocked for 1 h at 25° C. in 5.0% BSA (dPBS). The blocking solution wasremoved, and the wells were washed twice in dPBS. Recovered cells wereadded to the coated wells in a volume of 100 μl per well and placed at37° C. for 30 minutes (5% CO₂). Following this incubation, the assayplate was inverted and assay wells were washed twice with 100 μl dPBS toremove non-specifically adhered cells. The wells were then fixed inZamboni fixative, stained with Gill 1 hematoxylin, and overlayed with80% glycerol. The assay was imaged at 100× magnification, capturing 3random fields/well on a Zeiss Axiovert 200 inverted microscope equippedwith a Zeiss Axiocam digital camera. Readings represent the number ofadherent cells/well (average six high powered 100× fields, three takenfrom each of two separate wells).Adhesion Assays on Stainless Steel DisksAdhesion assays on 6 mm stainless steel disks (Orbus MedicalTechnologies, Ft Lauderdale, Fla.) pro-coated with anti-CD34 antibody,tropoelastin, coating vehicle, or uncoated metal were performed in asimilar fashion as described above, with the following modifications:48-well plates were used to hold the disks during the assay period tofacilitate manipulation of the disks and twice the volume of cellsuspension was applied (200 μl/well). Exemplary amounts of total proteincovalently attached to each disk were approximately 100 micrograms.Post-adhesion, the wells were washed twice in dPBS, using a swirlingmotion to dislodge non-specifically adhered cells. Wells were fixed for10 minutes at 25° C. in 4% neutral buffered formalin, washed three timesin dPBS, permeabilized in 0.5% Triton X-100 (dPBS), blocked in 5% nonfatmilk (dPBS), and stained for actin fiber assembly with Oregon Green488—conjugated phalloidin (Molecular Probes/Invitrogen, Carlsbad,Calif.). The disks were mounted to modified microscope slides withVectashield mounting medium+DAPI (Vector Laboratories, Burlingame,Calif.) and imaged under a mercury lamp on a Zeiss Axioplan 2 microscopeequipped with a Zeiss Axiocam digital camera.Migration Assays

Briefly, 16 h before the assay, 70% confluent 75 cm² flasks of humanmelanoma A2058 cells (ATCC, passage 3-12) or human aortic endothelialcells (HAEC; Cambrex, passage 4-7) were kept in basal media (dMEM+0.1%BSA, or EBM-2+0.1% FCS, respectively). Cells were lifted with 0.25%Trypsin/1 mM EDTA, diluted in 0.5% BSA (dMEM) and washed 3 times inbasal media (dMEM+0.1% BSA) or (EBM-2+0.1% FCS,) and recovered insuspension at working density (2×10⁶/ml for A2058 or 1.5×10⁶HAEC) for 1h at 37° C. (5% CO₂). For migration-inhibition assays, the cells werepreincubated during the recovery period for 30 minutes in 25 μg/mlanti-integrin antibodies (Chemicon, Temecula, Calif.) or control IgG(Jackson ImmunoResearch, Inc.). All migration assays were performed intriplicate in a 48-well Boyden chamber apparatus (NeuroProbe, CabinJohn, Md.). The recovered cells were added to the bottom chambers in avolume of 30 μl. The wells were overlayed with an 8 μm-porepolycarbonate membrane (NeuroProbe, Cabin John, Md.) and coated with 100μg/ml Rat tail collagen (Trevigen, Gaithersburg, Md.) for A2058 cells oracetylated 1% gelatin from porcine skin (Sigma, St. Louis, Mo.) forHAEC. The apparatus was assembled and stored inverted at 37° C. (5% CO₂)for a 2 h adhesion-period. The apparatus was re-inverted and 52 μl ofthe chemoattractants, 10 mg/ml Fibronectin, 200 ng/ml Tropoelastin, 10μM elastin-derived peptides; VGVAPG (SEQ ID No. 5), andIFPGGACLGKACGRKRK (SEQ ID No. 6) (Cterm peptide) or 0.1% BSA/dMEM(diluent, random migration control), were added to the upper chambersand the migration was allowed to proceed for 2 h in a humidifiedincubator at 37° C. (5% CO₂). The membranes were then removed, fixed inmethanol, stained with a Hema 3 stain set (Fisher Scientific,Pittsburgh, Pa.) and mounted migrated-side down to a 50×75 mm glassslide. Before 90% mounting medium (in xylenes) was applied, thenon-migrated cells were removed from the exposed, non-migrated face ofthe membrane with a moistened swab. Readings represent the number ofcells migrating through the membrane (the sum of two high power×20fields/well, averaged for each sextuplicate-well set).

CD34+ Cell Purification

Human CD34+ cells were purified from a peripheral blood mononuclear cellpreparation collected by an apheresis procedure. The donor was mobilizedwith 10 μg/kg granulocyte colony-stimulating factor (G-CSF) for fourdays prior to the collection procedure, as approved by the InstitutionalReview Board of the University of Utah. Purification of CD34+ cells wasaccomplished in a closed and sterile system using an automated cellselection device, CliniMACS (Miltenyi Biotec, Auburn, Calif.). Targetcells were labeled with super-paramagnetic particles comprised of ironoxide and dextran conjugated to murine CD34 monoclonal antibodies. Afterlabeling, the cells were separated using a high-gradient magneticseparation column. The magnetically labeled cells were retained in themagnetized column while the unlabeled cells passed through. The CD34+cells were recovered by removing the magnetic field from the column andwashing the cells out into a collection bag.Analysis of CD34+ enrichment and purity was accomplished by flowcytometry using a FACScan analyzer (Becton Dickinson, San Jose, Calif.).Cells were stained with the monoclonal antibodies CD45-FITC and CD34-PE(Becton Dickinson, San Jose, Calif.) in addition to the viability probe7-amino actinomycin-D (Sigma-Aldrich, St. Louis, Mo.). Briefly, dataanalysis was performed by gating on all leukocytes (CD45 positive cells)and all viable (7-AAD negative) cells to examine the CD34-PE expressioncompared to an isotype (IgG_(2a)-PE) control. At least 100,000 viableleukocytes were analyzed to determine the percentage of CD34 positivecells. Cells used for the experiment were 98% CD34+ cells.Recombinant Human TropoelastinThe endogenous signal sequence was removed and replaced with a10×-histidine tag. This cDNA was cloned into anIsopropyl-β-D-Thiogalactoside (IPTG)-inducible construct (pET System,Novagen/EMD Biosciences, San Diego, Calif.) and expressed in the E. colihost strain Rosetta-2 (DE3) pLysS (Novagen). LB growth media containing50 g/ml ampicillin and 34 μg/ml chloramphenicol were inoculated withRosetta-2 (DE3) pLysS bacteria containing the recombinant tropoelastinplasmid, and the culture was grown at 37° C. until optical density at600 nm was 0.4-0.6. Induction was accomplished by adding IPTG to a finalconcentration of 1 mM, and the culture was incubated at 37° C. for 4 b.The bacterial pellet was harvested by centrifugation at 16,000×g for 20min at 4° C. The pellet was resuspended in Bugbuster HT+lysozyme, andinclusion bodies were purified by washing (a total of 4×) with Bugbusterdiluted 1:10 with deionized H₂O followed by centrifugation at 4o at5,000×g. Following the last wash, the inclusion bodies were harvested bycentrifugation at 16,000×g for 15 min at 4° C. Inclusion bodies wereresuspended in 1× Bind Buffer (500 mM NaCl, 20 mM Tris-HCl, 5 mMimidazole) containing 6 M urea that had been adjusted to pH 7.9(following addition of the urea). Pellet was allowed to dissolve at 4°C. overnight. Insoluble material was then removed by centrifugation at16,000×g for 30 minutes. The supernatant was filtered through a 0.45 μmsyringe filter. The sample was then added to His-Mag beads (Novagen)that had been pre-equilibrated to 1× Bind Buffer with 6M urea and placedon a rocker for 5 minutes. Beads were collected using a Magnatight stand(Novagen) and washed×4 with 1× Wash Buffer (500 mM NaCl, 60 mMimidazole, 20 mM Tris-HCl) containing 6M urea (adjusted to pH 7.9).After the final wash, protein was eluted from the beads with 500 mMimidazole, 500 mM NaCl, 20 mM Tris-HCl, 6M urea, pH 7.9. The sample wasthen dialyzed against HBSS using Slide-A-Lyzer Dialysis Cassettes with10,000 MWCO (Pierce). Obtained protein was analyzed by SDS-PAGE analysisby standard techniques and stored at −80° C.Other ReagentsHuman collagen I and fibronectin for adhesion experiments, werepurchased from Biomedical Technologies Inc. (Stoughton, Mass.).Elastin-derived peptides were synthesized at the DNA/Peptide Facility,University of Utah.

REFERENCES

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All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

EQUIVALENT

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The invention claimed is:
 1. An implantable medical device coated with afragment of human tropoelastin, wherein the human tropoelastin fragmentconsists of the amino acid sequence of SEQ ID NO:
 6. 2. The device ofclaim 1, wherein the human tropoelastin fragment is covalently attachedto the device.
 3. The device of claim 1, wherein the human tropoelastinfragment is in an amount effective to promote adhesion of endothelialcells to the device.
 4. The device of claim 3, wherein the endothelialcells are endothelial stem cells or endothelial progenitor cells.
 5. Thedevice of claim 3, wherein the amount of the human tropoelastin fragmentis at least 100 ng per square centimeter.
 6. The device of claim 1,comprising a metal, an alloy or a plastic.
 7. The device of claim 6,wherein the metal comprises stainless steel.
 8. The device of claim 1,further comprising a biocompatible polymer.
 9. The device of claim 8,wherein the biocompatible polymer is selected from the group consistingof dacron, polyurethane, polypropylene, or polytetrafluoroethylene(PTFE).
 10. The device of claim 1, further coated with an anti-CD34antibody.
 11. The device of claim 10, wherein the anti-CD34 antibody ismonoclonal.
 12. The device of claim 1, wherein the device is a catheter,stent, shunt, wire, or other intraluminal device.
 13. The device ofclaim 1, wherein the device is a pacemaker, cardioverter-defibrillator,artificial valve, or vascular graft.